Techniques in Protein Chemistry
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
CHEM 108
Page 1
Spring 2020 version
TECHNIQUES IN PROTEIN CHEMISTRY:
AN INTRODUCTION
UCSD CHEM 108
SPRING 2020
Department of Chemistry and Biochemistry
University of California, San Diego
Thomas E. Crowley, Florence F. Davidson,
Stuart F. Duffy, Mark S. Johnson,
Jack Kyte, Charles L. M. Lumba
Contents
Legends for Cover Figures .................................................................................................................... 2
Abbreviations ...................................................................................................................................... 2
Biochemistry Laboratory Safety ........................................................................................................... 3
Chemistry Laboratory Regulations ....................................................................................................... 3
Protein Handling Guidelines................................................................................................................. 7
Exercise 0 Laboratory Introduction: Pipetting practice and the Spec20 spectrophotometer ............... 10
LDH Section I: Purification and Characterization of Lactate Dehydrogenases.......................................... 17
Introduction ...................................................................................................................................... 17
Exercise 1 Preparation of Isoenzymes of LDH from Tissue Extracts ..................................................... 20
Exercise 2 Enzymatic Assay of LDH by Spectrophotometry ................................................................. 24
Exercise 3 Kinetic Analysis of LDH ...................................................................................................... 31
Exercise 4 Separation and Visualization of LDH Isoenzymes by Electrophoresis on Cellulose Acetate . 39
Exercise 5 Purification of LDH by Precipitation with Ammonium Sulfate ............................................. 45
Exercise 6 Purification of LDH by Affinity Adsorption to BioSepra Blue Trisacryl MR ........................... 50
Exercise 7 Protein quantification assays: Bradford and UV-absorption assays .................................... 56
Exercise 8 Electrophoresis of Native Proteins on Polyacrylamide Gels ................................................ 61
Exercise 9 SDS-PAGE of the Initial Supernatant and Purified LDH ....................................................... 72
Exercise 10 Western Blot ................................................................................................................... 78
LuxG Section II: Expression, purification, and characterization ............................................................... 84
Introduction ...................................................................................................................................... 84
Exercise 1 Expression of LuxG-his6 in E. coli and Release by Lysis ....................................................... 92
Exercise 2 Affinity Purification of LuxG-his6 with Nickel Column......................................................... 96
Exercise 3 SDS-PAGE of Tuner/pGhis Lysate and Purified LuxG-his6 ................................................... 99
Exercise 4 Flavin Reductase Activity Assays ...................................................................................... 103
Appendix ............................................................................................................................................. 106
Derivation of Lineweaver-Burk equations for competitive, noncompetitive, and uncompetitive
inhibition ......................................................................................................................................... 106
Reagents for Polyacrylamide Gel Electrophoresis (PAGE) ................................................................. 109
Operation of the Spectronic 20D+ (Spec 20)..................................................................................... 110
Preparation of an Acrylamide Gel for Electrophoresis ...................................................................... 112
Absorbance Measurements with Multiwell Plates and an Automated Plate Reader ......................... 114
Increasing the Concentration of Protein with a Centrifugation/Filtration Device .............................. 116
Table of Contents
Legends for Cover Figures
Left: The association of the riboflavin substrate with the E. coli flavin reductase Fre. The redox cofactor,
NADH, binds to the right side of the structure. Ingleman, M. et al. (1999) Biochemistry 38: 7040-7049.
Right: The association of NADH with the cofactor binding site of human LDH. The enzyme’s substrate,
pyruvate, is shown close to the nicotinamide ring of NADH, near the bottom of the enzyme’s structural
schematic. Chaikuad, A. et al. (2005) Biochemistry 44: 16221-16228.
Abbreviations
A
L
M
cm
BME

g
l
mA
mM
mg
mL
mm
ng
nm
rpm
s
M
mol
g
L
absorbance
liter
molar
centimeter
Beta mercaptoethanol
extinction coefficient
gram
path length
milliampere
millimolar (10-3 molar)
milligram
milliliter
millimeter
nanogram (10-9 gram)
nanometer
revolutions per minute
seconds
micormolar (10-6 molar)
micromole
microgram
microliter
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Biochemistry Laboratory Safety
Unlike some undergraduate laboratories that you have probably encountered, an undergraduate
biochemistry laboratory uses few potentially explosive reactions and caustic solutions. Because of the
insidious nature of some hazards used in this course, however, you should always be aware of the
hazards of what you are working with. For instance, in one of the experiments you will be handing a
neurotoxin (acrylamide). Due to the hazards of this reagent, do not ever mouth pipette it and always
wear gloves. Another reagent you may come across is barbital buffer which contains a barbiturate, a
central nervous system depressant. Similar mindfulness should be used when you come across this
reagent. Always use a pipette or a pipette bulb.
Pipetting: If you have not used measuring pipettes or safety bulbs before, make sure you obtain a
demonstration from your teaching assistant. Pipetting is a basic technique used extensively in all areas
of biochemistry, not only in this laboratory.
For pipetting, keep these points in mind.
• Use the correct pipette, for example check whether you are using a blow-out pipette or nonblow-out pipette.
• Never draw liquid up into the bulb; this contaminates both the bulb and the solution you
are using. Practice pipetting with water until you are proficient.
• Avoid parallax; hold the pipette up so that the meniscus is seen from the side as a single line
and read the meniscus at the bottom.
Chemistry Laboratory Regulations
Prepare carefully
Attentive and considerate behavior is expected at all times. Maintain clean laboratory benches and
common areas. Clean your own work area, any common areas where you have worked and any area
assigned to you by your TA.
EATING, DRINKING, GUM CHEWING, AND SMOKING ARE FORBIDDEN in lab (to avoid chemical ingestion,
excessive inhalation of harmful vapors, and ignition sources). Food, drinks, and smoking materials
(including electronic cigarettes) should be left outside the lab or stored in securely closed containers
and left in the storage cabinets located near the entrance of the laboratory.
Prepare & Protect Yourself
Listed is the minimum level of safety protection necessary to work in the Undergraduate Teaching Labs:
safety eyewear, long pants or skirt, closed-toed shoes, and long (knee-length) lab coat. Skin between
the shoes and pants/skirt should be covered. Students who arrive unprepared or inappropriately
dressed will be dismissed until ready to work.
SAFETY EYE PROTECTION: Eye Protection must be worn by everyone at all times. All students, faculty,
staff, and visitors are required to wear approved safety glasses or splash goggles in addition to any
prescription glasses.
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Chemical splash goggles are required whenever anyone transferring more than a small amount (~25 mL)
of a hazardous material or when performing an operation involving a splash hazard. Safety glasses are
designed for use in normal laboratory operations but offer minimal splash protection.
Approved glasses and goggles are available at the UCSD Bookstore in the Price Center and the General
Store Co-op in the Student Center. If you wear prescription glasses, choose safety glasses designed to fit
over your glasses or choose goggles. The Undergraduate Labs Stockroom does not lend or sell goggles or
glasses to students.
Additional eye and face protection (e.g., full-face shields) are available and should be used as directed
by the experimental procedure or the lab supervisor, or when the level of eye hazard is unknown.
Always be sure to use proper eye protection when ultraviolet (UV) lamps or lasers are used.
Contact lenses: The current understanding is that using contact lenses in lab creates no additional
hazard. When worn with the safety eyewear required of everyone, contact lens wear is acceptable.
APPROPRIATE CLOTHING: Lab coats, long pants or skirt and closed-toed shoes are required in the labs.
Choose sturdy shoes and a long style (knee-length) lab coat. Button the coat closed to protect skin and
clothing; make sure shirt sleeves don’t protrude beyond coat sleeves. Avoid loose or very long clothing;
remove loose jewelry; secure long hair and loose clothing away from flames, equipment, and chemicals.
GLOVES are provided in the labs and should be worn when working at all times. Protective gloves are
made with different materials. When working with hazardous chemicals you should know which
materials are compatible with the chemical you are handling. For this class, nitrile or latex gloves will
suffice. Ask the lab staff if you do not find suitable gloves stocked in your classroom (usually stored
under the chalkboard). Change your gloves often and remove them and wash your hands before leaving
the lab and entering public areas.
KNOW THE HAZARDS OF MATERIALS before you begin any procedure. Check the appropriate sections in
the lab manual for guidelines.
MATERIAL SAFETY AND DATA SHEETS (MSDS) are available in the laboratories, in the Science &
Engineering Library, and in the Undergraduate Chemistry Lab Stockrooms (YORK 3150 and NSB 1104)
and at the website www.ucmsds.com.
KNOW EMERGENCY PROCEDURES AND SAFETY EQUIPMENT: In each lab, note exits and evacuation
routes, presence/absence of installed telephone, location of first aid station(s), shower/eyewash
stations, spill control materials, and fire extinguishers. Know how to summon assistance from the
Stockroom, Campus Police, or EH&S, as appropriate. (Contact information below)
KNOW YOUR OWN LIMITS: If you have limited mobility or any condition that may limit your ability to
work safely, consult with the lab staff, campus EH&S, and your health care provider. If you carry
medication that might be needed on an emergency basis (e.g. for diabetes or asthma), inform your lab
supervisor. Work stations for physically impaired or temporarily disabled students are available; if you
need these facilities, ask your Instructor. Emergency Response Information Forms allow students & staff
to communicate medical information to emergency responders; blank forms are available in the lab
Stockrooms. Fill these forms out, fold them, and adhere them to the inside cover of your lab notebook.
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Prevent Accidents
Horseplay and pranks are especially dangerous in a laboratory setting and are forbidden at all times.
Keep all lab materials away from the face and mouth. Never pipet or start a siphon by mouth; this has
been a source of serious laboratory mishaps. Never work alone in the laboratory and never perform
unauthorized experiments. Students are to be in the Undergraduate Labs only when attended by an
Instructor, TA, or member of the lab staff.
HAZARDOUS MATERIALS HANDLING: Label all containers with their contents and concentrations. Store
hazardous materials away from unsecured ledges for increased safety in the case of an earthquake or
other extreme condition. Use secondary containers (trays or tubs) and segregate materials according to
hazard classes (e.g. don’t store acids with bases). Store hazardous materials below eye level and return
all materials to their proper storage locations. Date containers when first opened.
Special secondary containers are provided for carrying hazardous materials outside the lab or between
labs. To obtain a refill from the Stockroom, choose the appropriate secondary container to carry the
empty bottle to the Stockroom. Request a refill and carry the filled container back to lab in the
secondary container. Return the filled bottle and the carrier to their proper storage locations.
HAZARDOUS WASTE MANAGEMENT: Hazardous waste should be disposed of in the provided
containers. Choose the correct container for chemical hazardous waste and for all broken glass and
other sharps. Unless explicitly instructed, do not dispose of any waste in the drains. Read labels and the
lab manual and ask the TA, the Instructor, or the lab staff for your course for waste disposal guidelines.
The Environment, Health and Safety Specialist at the Undergraduate Labs (see below) or the lab safety
staff at UCSD EH&S (x 43660) can also help you find information.
Use fume hoods for all work involving or producing flammable, corrosive, or fuming chemicals (i.e.,
ammonium hydroxide or other strong acids and bases). Any volatile toxic substance should be opened
and used only in a hood. Examples of volatile chemicals used in this lab are TEMED and mercaptoethanol (2-mercaptoethanol, ME, 2ME)
Respond Appropriately to Accident, Emergency, or Sudden Illness
SUMMON ASSISTANCE and – if you are trained – ADMINISTER FIRST AID. Call the Undergraduate Lab
Stockroom (see below) or send an uninjured person with a message. TAs must not leave students
unattended in the classroom. Emergency contact information is posted near each telephone. If you
suspect an ambulance will be needed, do not hesitate to call 9-1-1 (534 – HELP from a cell phone) for
assistance.
An ACCIDENT REPORT is necessary for any accident or chemical spill, no matter how minor the incident
seems. These records are important in identifying recurring injuries, near misses, or problem areas.
PERSONAL EXPOSURE: If clothing catches fire or if a hazardous chemical is spilled on skin or in eyes,
assist the exposed person to the shower/eyewash and rinse the areas of contact with copious amounts
of water for 15 minutes or until assistance arrives; remove contaminated clothing. Call 9-1-1 or send an
uninjured person to notify the lab staff to ensure injuries receive proper treatment.
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Do not attempt SPILL CLEAN-UP without proper personal protective equipment (PPE). For large or very
hazardous spills, call 9-1-1 for assistance. For small spills, use the spill clean-up kits and PPE provided;
consult your lab supervisor and Material Safety Data Sheets for clean-up precautions. Double bag and
label contaminated materials and store in the Hazardous Waste Area of the lab. Notify the lab staff so
that disposal can be arranged.
For a mercury (Hg) spill, use only the mercury collectors provided in the spill kits. Do not mix mercury
with any other waste. There are no waste handlers that will accept such mixed waste.
BUILDING EVACUATION: In any emergency situation, assure the safety of people before considering any
damage to property. When instructed, leave the lab immediately. Use stairs, never elevators (power
may fail in an emergency). Pull the fire alarm as you exit. As soon as you reach a safe location, call 9-1-1
(534 – HELP from a cell phone) and report the situation to the UCSD police. Go to the assigned assembly
location for your lab. The lab supervisor will take attendance (to assure everyone is safe) and provide
this information to responding emergency personnel. Do not leave the area or reenter buildings until
instructed to do so. Note any injuries to yourself or others and any remaining dangers. Provide
assistance to injured persons, as long as you do not place yourself in additional danger.
FIRE: Do not attempt to fight fires in the lab (except on clothing, in which case use shower or fire
blanket); evacuate the lab quickly (see Building Evacuation), closing all doors. Call for assistance when in
a safe location. Fire extinguishers are placed in the labs for use on small fires by trained personnel
working in pairs.
EARTHQUAKE: Move away from overhead lights, heavy unsecured objects, and hazardous materials.
Choose a sheltered position to wait (under a table, in a door frame, or against a bearing wall). Once the
tremor stops, shut down gas lines & heat sources. Exit the building quickly (see Building Evacuation).
CHEMISTRY & BIOCHEMISTRY UNDERGRADUATE TEACHING LABS York Stockroom (YH 3150): 534-0222
NSB Stockroom (NSB 1104): 822-4316 Chemistry & Biochemistry Dept. Safety Director: John Palmer, PhD
(534-5906)
Report all emergencies to campus police: 9-1-1, (534 - HELP from a cell phone)
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Protein Handling Guidelines
Throughout this class you will be handling proteins. Proteins are generally delicate molecules. There
are some notable exceptions that are capable of maintaining biological activity despite facing harsh
treatment. For example, RNase can maintain its activity despite being subject to temperatures up to
100oC and denaturation by detergents. Since RNase can be found in perspiration or made by bacteria
found on hands, RNase contamination is a constant worry for scientists studying RNA. Another example
is prions. Prions are proteins that are capable of causing disease, the most notable being the protein
that causes bovine spongiform encephalitis (BSE or commonly known as mad cow disease). Scientists
who work with prions must be very careful when handling their protein samples since the infectious
form of these proteins are extremely stable to even the most physically and chemically harsh conditions.
Nonetheless, most proteins are fairly fragile. Enzymes, for example, tend to readily lose activity even in
their native environments. If they did not, then we wouldn’t need to eat so much to obtain the
nutrients and energy to replace them. For the vast majority of biochemists, great lengths are
undertaken in order to preserve their protein of interest’s activity so that it can be accurately
quantitated and studied. Most of the techniques described in this section are common methods used to
preserve a protein, preventing it from denaturing, or losing its native structure. Consider that most
proteins have been fine-tuned from billions of years of evolution. It is therefore conceivable that the
smallest levels of denaturation can have negative effects on a protein’s function.
Keep protein samples cold
Keep all samples that contain your protein of interest on ice. Samples include tissues, extracts, and
solutions containing proteins. For long term storage, place the samples in the refrigerator or freezer,
according to the sample’s tolerance. High temperatures allow weaker hydrophobic interactions in the
protein to become disrupted, thereby causing the protein to denature, or lose its native structure, and
become inactive. Besides preventing thermal denaturation, low temperatures decrease the activity of
proteinases that may be present. Additionally low temperatures inhibit growth of microorganisms
which will happily metabolize proteins.
Use Pure Water
The quality of the solvent, water, used in the preparation of all solutions and the final rinsing when
cleaning glassware is crucial. Tap water should never be allowed to come into contact with proteins.
Tap water contains large amounts of ions which may have undesired effects on proteins. Additionally,
an appreciable amount of organic solvents and heavy metals can be found in tap water which can serve
to denature proteins. House “distilled water,” though more pure, is processed by an ion
exchange/filtration procedure and usually contains organics; its use is also discouraged. Ideally, the
water would be glass distilled for use. Since the facilities for large scale distillation are not available, we
have a filtration system in the stockroom that provides water pure enough for biochemistry. The 20-liter
carboys near the sinks in the lab contain water from this system.
Detergents and Soaps. Sometimes contaminants are inadvertently added. Detergents and soaps are
common cleaning agents for labware, however contamination by these two agents is to be avoided at all
costs since they can interact with proteins and cause denaturation. Fortunately these cleaning agents
can usually be rinsed away with copious amounts of water. For cleaning your labware, use the following
procedure:
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a. Clean with a solution of detergent.
b. Rinse three times with tap water.
c. Rinse three times with house “distilled water.”
d. Rinse three times with the purified water from the carboys.
Three rinses at each step is the minimum necessary to achieve thorough rinsing – remember this when
it comes time to rinse a cuvette or other container with buffer before use.
Metal items (e.g. scalpels, scissors, and tweezers) should be rinsed with 70% isopropanol before storage
to prevent rusting.
Avoid Heavy Metals
The cations of heavy metals such as lead, mercury, and several others can rapidly bind to and inactivate
a protein. Therefore, breaking a mercury thermometer is a potentially serious source of contamination
for both the experimenter and the experiment and requires consultation with the instructor with regard
to appropriate cleanup and disposal.
Avoid organic solvents
Proteins usually have a hydrophobic core and a hydrophilic exterior. This structural arrangement is
stabilized in water where the hydrophilic exterior forms hydrogen bonds with highly polar water
molecules. Organic solvents have less polar character than water and will destabilize a protein’s
structure by making hydrophobic interactions at the protein’s exterior more favorable, promoting the
protein to turn inside-out. Due to their denaturing effects, organic solvents should be avoided.
Limit Oxidation from air
Intracellular enzymes, especially those with an essential cysteine, can sometimes be inactivated by
oxidation. This oxidation can be prevented by the addition of reducing agents such as 2mercaptoethanol (-mercaptoethanol/ME/2ME), glutathione, or dithiothreitol (DTT). These agents are
rarely used, however, with extracellular proteins where cysteines are normally in their oxidized form of
cystine. Additionally, careful consideration must be made before these agents are used since they may
interfere with procedures such as the Lowry method for protein determination or the Ni affinity
purification.
Limit Foaming
If you have ever prepared a meringue for a pie or added egg whites to a margarita for froth, you have
observed the effects of the denaturation of the proteins in the egg white (predominantly ovalbumin).
Protein readily denatures at a surface between air and water and, after it has denatured, it becomes a
surfactant similar to soap or detergent. Although a little bit of foaming is not serious and often helps you
to locate your protein, a lot of foaming will denature your protein significantly alter the protein’s
activity. Avoid foaming. It is often caused by overzealous agitation or stirring or solutions. In this regard,
you should be aware that the speed of electric stir motors in the cold room increases dramatically as
they warm up. With concentrated solutions of protein, foaming and bubbles are a particular problem
when transferring the solution by Pasteur pipette. To avoid this problem, try not to draw more air into
the pipette than is necessary.
Prevent Fungal and Bacterial growth.
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Growth of bacteria and fungi can be kept at a minimum by keeping solutions of proteins cold, sterilizing
scalpels and scissors with isopropanol, and using gloves when handling dialysis tubing. A few drops of
isopropanol or toluene have been added as preservatives to some of the reagents used in this course
(e.g. the slurries of chromatography resins: AMP-Sepharose and Sephacryl S-300). Washing the columns
before you use them removes these organic solvents.
Some biochemists use sodium azide or thimerosal to solutions to prevent them from fouling. However,
these reagents are toxic to humans as well as to microbes so they should be used with caution.
Interestingly, thimerosal is a mercury compound that is used to preserve some vaccines.
pH
Enzymatic activity of proteins is usually affected by changes in pH, and proteins are unstable and
denature at extremes of pH. The behavior of proteins on chromatography and electrophoresis is
affected by variations in pH as well. It is for all of these reasons that a buffer is used in nearly all
solutions containing protein to maintain the pH. Buffers, however, have only a finite capacity for
absorbing or releasing protons to keep the pH constant. There is always a possibility that one will
inadvertently add acid or base in excess of the capacity of the buffer. Therefore, it is a good idea to
periodically check the pH of your solution of protein, certainly before a major step such as measuring
enzymatic activity or loading it onto a chromatographic column or electrophoretic separation. The
easiest way to do this is to place a microliter of the solution on a strip of an appropriate pH paper.
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Exercise 0 Laboratory Introduction: Pipetting practice and the Spec20
spectrophotometer
Key principles
Pipetting, Beer-Lambert Law, spectrophotometry
Figure 1 Pipettes. You will be given a set of 3 pipettes. A. They are identified as P1000, P200, and P20 by the labels located at
the top of each pipette. B. Before use, the pipettes should be fitted with pipette tips. The P1000 uses the blue tips whereas the
P200 and P20 use the yellow/white tips. C. The setting window is different for each pipette. The setting is adjusted by twisting
the black knob (arrow) which sets the volume (in microliters, L) that will be dispensed. The P1000 (left) has one red number
which represents the thousands place unit. For example, the setting shown in the figure for the P1000 indicates 1000 L or 1
mL will be transferred. The P200 (center) has no red digit. The setting for the P200 simply represents the volume in microliters
that will be dispensed. The setting shown in the figure indicates that 200 L will be transferred. The P20 (right) has one red
digit which represents the tenths place unit. The setting shown in the figure indicates that 2.5 L will be transferred.
In this lab, you will be introduced to using the lab pipettes (see Figure 1). There are three pipettes that
you will use throughout the class, a P1000, P200, and P20. Each pipette can be adjusted to transfer the
desired volume of liquid by twisting the black knob, visible from the side of the pipette, pointed out by
the arrow in Figure 1. The P1000 should be used to pipette between 200 L and 1000 L. The P200’s
range is between 20 L and 200 L. The maximum volume for the P20 is 20 L. It is important to never
attempt to set the pipette to a value greater than the maximum volume or a value below zero. Doing so
damages the pipette.
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Figure 2. Using a pipette. A. Adjust the setting, attach the appropriate tip to the end of the pipette. B. Press the plunger down until you feel clear
resistance. This is the “first stop.” Place the tip below the surface of the liquid you will pipette. C. Slowly lift your thumb, releasing the plunger. If
the plunger is released too quickly the buffer may be drawn up the pipette shaft causing contamination. D. Place the pipette tip into the new
vessel; then press the plunger down to the first stop. E. Some liquid will remain in the tip. To expel the remaining liquid, press the plunger down
past the first stop, to the “second stop.” A bubble will form at the end of the pipette tip (arrow). F. Dispose of the tip by holding it over the waste
container and pressing the tip ejector button.
The procedure for using a pipette to transfer a volume of liquid is presented in Figure 2.
Common mistakes include:
1. Pressing the plunger passed the first stop and then aspirating. This will draw up too much liquid.
2. Not touching the pipette tip to liquid or a tube surface while dispensing. This tends to cause
some of the liquid to remain ON the pipette tip. Too little liquid will be transferred.
3. Not pressing the plunger down to the second stop for dispensing. This tends to cause some of
the liquid to remain IN the pipette tip. Too little liquid will be transferred.
4. Releasing the plunger before raising the pipette tip from the liquid. This causes some of the
liquid to get drawn back into the pipette tip. This effectively removes some of the liquid that
was transferred and reduces the volume of liquid in the tube by some random amount.
For this experiment you will be practicing proper use of the pipettes to accurately measure small
volumes. You will also take this opportunity to gain experience measuring absorbance and familiarize
yourself with the spectrophotometer.
Absorbance is a unitless value (i.e. not measured in mole, g, mg/mL, molar, etc.) that is measured with
an instrument known as a spectrophotometer. The wavelength of light is chosen by the user and a set
intensity of light is directed through a sample (incident light). The light that is not absorbed by the
sample (transmitted light) is measured by a detector. The output reading given by the
spectrophotometer is absorbance which is defined as follows:
Techniques in Protein Chemistry
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𝐼1
𝐼0
𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = −𝑙𝑜𝑔 ( ) = 𝑙𝑜𝑔 )
(
𝐼0
𝐼1
I1 = transmitted light
I0 = incident light
Page 12
Fall 2018
Eq. 0-1
Take a moment to consider what an absorbance reading actually means. As you continue through this
course, you will be taking many absorbance readings, and it is important to keep in mind what you are
actually measuring as well as the limitations of the instrument. What is the expected absorbance
measurement if the sample absorbs no light? If the sample absorbs no light, then I 1 = I0 so absorbance
will equal zero. How much of the light would have to pass through the sample in order to obtain an
absorbance reading that is less than zero? With this in mind, would you consider an absorbance reading
that is less than zero reliable? At the other extreme, what is the absorbance reading if 99% of the light is
absorbed by the sample and only 1% is transmitted? In this case, I 1/I0 = 0.01 and the absorbance reading
will be two. Would you consider an absorbance reading of two reliable? The answer is probably not.
The detector that is measuring transmitted light would have to be very sensitive to make this
measurement accurately.
Newer spectrophotometers may have more sensitive detectors that can accurately measure higher
absorbance values. One should keep in mind, though, that high absorbance values are often less
reliable. Other instruments (e.g. a nanodrop) reduce the path length of the sample to measure samples
with a high absorbance value. A multiplier value is used to adjust for the smaller path length. With
these instruments, small errors in the instrument’s reading is amplified by the multiplier.
Many chemical compounds absorb light at particular wavelengths while in solution. This absorbance is
directly proportional to the concentration of the absorbing molecule according to the Beer-Lambert Law
(Eq. 0-2).
𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 =  ∗ 𝑐 ∗ 𝑙
 = extinction coefficient, a constant
c= concentration of absorbing molecule
l = path length that the light through the sample (dependent on the
sample container and almost always 1 cm)
Eq. 0-2
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Figure 3 Results of an absorbance scan of bromophenol blue in phosphate buffered saline (PBS). The x-axis
represents wavelength in nm and the y-axis is absorbance. The absorbance maximum is ~591nm.
In this lab, you will be monitoring bromophenol blue, which is a pH indicator that is blue at high pH. If
the absorbance of a solution of bromophenol blue is measured over a series of wavelengths, there will
be an absorbance maximum at 591 nm (Figure 3). Selecting the absorbance maximum allows maximum
sensitivity for measuring the amount of molecule since smaller amounts of the absorbing molecule will
have the best chance of being detected at this wavelength.
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Absorbance of bromophenol blue
1.5
1.3
A591nm
1.1
y = 0.1428x + 0.0323
R² = 0.996
0.9
0.7
0.5
0.3
0.1
-0.10.00
5.00
10.00
15.00
g/mL
20.00
25.00
30.00
Figure 4 Absorbance vs. concentration plot of bromophenol blue.
Since absorbance is directly proportional to the concentration of the absorbing compound, plotting
absorbance vs. concentration should show a linear relationship (Figure 4).
Materials:
In lab: P20, P200 and P1000 Pipetman, vortexing mixer, Eppendorf tubes, 13 x 100 mm glass culture
tubes, Parafilm, 0.25% (w/v) bromophenol blue diluted in phosphate buffered saline, Spec20
spectrophotometer
Safety: the solutions and tissues used in this lab are not toxic and can be disposed of down the drain.
Wear safety glasses and gloves at all times. Glass tubes should be disposed of in glass waste along with
graduated and Pasteur pipettes.
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Procedure:
Tube
1, 2, 3
4, 5, 6
7, 8, 9
10, 11 12
13, 14, 15
16, 17, 18
19. 20, 21
22, 23, 24
25, 26, 27
28, 29, 30
31, 32, 33
0.25% (w/v)
bromophenol bromophenol
blue (g/mL)
blue (L)
H2O to
make 3 mL
(L)
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Fall 2018
A591
A591
A591
0.00
2.08
4.17
6.25
8.33
10.42
12.50
14.58
16.67
18.75
20.83
Table 0-1
You will need to make 11 concentrations of bromophenol blue in triplicate. The concentrations are
provided in the second column of the table. Each sample will need to be 3 mL in order to be read in
the Spec 20. Calculate the volumes of bromophenol blue and water that are required to obtain 3 mL at
the appropriate concentration. Use Table 0-1 for assistance. Note 0.25% (w/v) = 0.0025 g/mL.
8)
1) Turn on the Spec 20 spectrophotometers to warm up the lamp (15 min)
2) Set up the Spec 20
a. Instructions are located on each instrument. Set the wavelength to 591 nm, set the filter,
and adjust the 0 and 100% transmittance. (Further information found in the Appendix)
3) Obtain 500 L of 0.25% (w/v) bromophenol blue solution and bring it to the bench.
a. Suggestion: Use a 1.5 mL Eppendorf tube to store the bromophenol blue.
4) Label thirty-three 13 x 100 mm glass culture tubes
a. These tubes are disposable and ink can be placed directly on the tubes
5) Measure the appropriate volumes of bromophenol blue and water into the culture
tubes according to Table 0-1.
6) Mix by vortexing or by sealing with Parafilm and inverting.
7) To take the absorbance reading
a. Insert the glass tube
b. Make sure the tube sits in the center of the holder
c. Close the black lid
d. Wait a few seconds to stabilize the reading
i. Some Spec 20s take as long as 15 seconds
Record the readings for each sample
Handling the Data
1) Calculate the average measurement for each bromophenol blue concentration.
2) Make a plot of absorbance vs. concentration
3) Calculate the regression equation for the LINEAR portion of the plot.
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
LDH Section I: Purification and Characterization of Lactate
Dehydrogenases
Page 16
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Introduction
Lactate dehydrogenase (LDH) is an enzyme that catalyzes the reaction:
CH3COCOO- + NADH + H+ ⇄ CH3CHOHCOO- + NAD+
(pyruvate)
(lactate)
In a given species of vertebrate, there are at least two unique forms of LDH. One form predominates in
more anaerobic tissue such as skeletal muscle (M-type), and the other predominates in aerobic tissue
such as heart (H-type). The bovine M-type is 332 amino acids while the bovine H-type is 334. Because
the two types of subunits can combine at random to form a tetramer, five different versions of LDH are
possible (M4, M3H, M2H2, MH3, and H4). Each version represents an isozyme of LDH since it catalyzes
the same reaction as LDH, but the amino acid sequences differ. The differences between the M-type
and H-type (the two isoforms) can be observed in an amino acid sequence alignment shown in Figure 1.
Most organs synthesize both isoforms but are predisposed to express more of one isoform than the
other. This ratio imbalance affects the distribution of each isozyme present in the particular organ. The
differences in amino acid sequence among isoforms causes them to possess distinct physical and
enzymatic kinetic properties. Expressing different isozyme ratios is one way cells can regulate enzymatic
activities according to their needs.
LDH: Introduction
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Figure 5. An alignment emphasizing the similarity of the amino acid sequences of bovine (Bos taurus) LDH isoforms. LDHA = muscle,
LDHB = heart, LDHAL6B = liver, LDHC= testis. Amino acids that are identical in all sequences are highlighted in black. Those that are
similar/highly conserved are boxed.
Selected References:
Everse, J., and Kaplan, N. O. (1973) “Lactate Dehydrogenase: Structure and Function,” Advances in
Enzymology, Vol 37 (Meister, A., Ed.) John Wiley and Sons, New York, pp. 61-133.
Everse, J., Zoll, E. C., Kahan, L., and Kaplan, N. O. (1971) Bioorganic Chemistry 1:207-233.
LDH: Introduction
Techniques in Protein Chemistry
CHEM 108
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Holbrook, J. J., Liljas, A., Steindel, S. J., and Rossman, M. G. (1975) The Enzymes, Vol XI (Boyer, P.D., Ed.),
Academic Press, New York, pp. 191-292.
Bovine LDH Gene and Protein Sequences in Databases
The gene and protein databases are critical tools for biochemists. In addition to sequences, they contain
literature citations and protein structure-function relationships. Searching for LDH entries is a good
introduction to the use of these sources. It will also provide relevant information for your reports.
Database searches for other proteins may be necessary for some of the exercises.
The skeletal muscle version is designated LDHA and the heart version LDHB. The liver and testis versions
do not need to be considered for this course.
---------------------------------UniProt Database----------------------------------------------www.uniprot.org
Search in: Protein Knowledgebase (UniProtKB)
Query = LDH, then order by organism, then page ahead and scroll to Bos taurus (bovine)
Should get:
Entry Name
Accession #
# amino acids
LDHA_BOVIN (skel muscle)
P19858
332
LDHB_BOVIN (heart)
Q5E9B1
334
LDHC (testis)
Q2KJG0
241
LDHAL6B (liver)
Q3T056
381
---------------------------------------NCBI Database-----------------------------------www.ncbi.nlm.nih.gov
<Search> drop-down menu in upper-left: select “Protein”.
Enter “LDH” in the search box
Many hits will be produced
Select “Advanced” to narrow the search
Search “All Fields” for LDH
AND search “Organism” for “Bos taurus”
Click <Search>
Should get:
gene name
LDHA (skel muscle)
LDHB (heart)
LDHC (testis)
LDHAL6B (liver)
Accession #
same as UniProt
same as UniProt
AAI05362
same as UniProt
gene ID
281274
281275
537256
509519
# amino acids
332
334
241
381
Some of results will be duplications. You can narrow the search results by selecting RefSeq which will
yield reference sequences. Results can be further refined by selecting UniProtKB/Swiss-Prot to crossreference with that database.
LDH: Introduction
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
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Exercise 1 Preparation of Isoenzymes of LDH from Tissue Extracts
Key Principles:
Proper protein handling technique. Homogenization to rupture cells and release cytoplasmic contents.
Centrifugation to separate cell components.
Background
For this laboratory period, you will be working with a partner to prepare material that you will be using
for your future experiments with LDH. As with any research topic, researchers need to obtain samples
in order to conduct experiments. Historically (and sometimes still applicable today), protein samples
were obtained from an endogenous source, meaning that the protein of interest was extracted from
tissues or organs that have been found to naturally express large quantities. If we wanted to study
blood clotting proteins, we might use blood tissue. If we wanted to study insulin, we might purify it
from the pancreas. Similarly, if we want to study LDH, we might try to obtain it from the liver, heart,
skeletal muscle, or testis since they are all known sources.
One point to keep in mind when obtaining proteins from endogenous sources is the location of the
protein within the tissues with which you are working. For example, can you think of how obtaining
blood clotting factors from blood or insulin from the pancreas might differ from obtaining LDH from
skeletal muscle? The answer is that blood clotting factors and insulin are extracellular molecules. They
are secreted outside of cells, making them readily available. On the other hand, LDH is a cytosolic
protein. It is not as accessible. Therefore one must break open or lyse the cells in order to obtain LDH.
Note that blood clotting factors and insulin are produced by cells, so you can obtain these proteins by
lysing appropriate cells. However, these secreted molecules tend to be processed with posttranslational
modifications before secretion. Therefore, lysing cells to obtain these proteins would enrich the
immature protein content in the preparation.
There are a number of methods to lyse cells. The method used often depends on the type of cell you
are attempting to disrupt. Most eukaryotic cells from the animal kingdom are relatively easy to lyse.
This is because they do not have cell walls like bacteria, plant, and fungi. In this lab, we will be using
mechanical force or shearing force generated by a blender and dounce homogenizer to break open the
cells. As you might surmise, these methods also generate heat which could serve to denature enzymes
such as LDH. It would be prudent for you to monitor the temperature of the homogenate to make sure
it doesn’t get too hot! Technically, these proteins normally function at 37oC, but also consider how
quickly steak breaks down at this temperature vs. in a 4oC refrigerator.
The time allotted for this lab is 4 hours. Can you imagine how much of a nuisance it would be if you had
to make an extract every time you needed to do an LDH experiment? Luckily proteins are relatively
stable at low and there are storage techniques that can help make your protein last indefinitely. The key
is temperature. Keeping your extract at 4oC (on ice) while you are working with it will help your extract
last. Similarly, you can store the extract in a refrigerator for short term storage just as you would store
many foods to preserve their quality.
Continuing with the food analogy, proteins stored in the freezer can last a very long time. Care must be
taken, however, since freezer temperatures (-10oC to -20oC) are not native temperatures for most
proteins. Continually freezing for storage and then thawing for experimentation will cause many
LDH Exercise 1: Preparation of Isoenzymes of LDH from Tissue Extracts
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proteins to lose activity/denature. To avoid freeze/thaw cycles, protein samples are often aliquoted,
meaning the total preparation is divided into smaller volumes. Then when an experiment needs to be
conducted, one aliquot is thawed and then discarded or stored in the refrigerator at the end of the day.
Ice crystals are similarly detrimental to proteins’ structure and activity. This effect is likely due to the
difference in the structure of H2O in ice vs. water. You may recall from general chemistry that one
unique feature of H2O is that the solid form (ice) is less dense than the liquid form (water). Ice actually
tends to expand upon freezing and this expansion is likely a contributing factor toward denaturing
proteins. Two techniques are often employed to prevent ice formation and avoid protein denaturation.
One method is to quick-freeze in a super-cooled solution of dry ice and isopropanol (~-78oC). The goal
for quick-freezing (also known as snap freezing) is to freeze small volumes at such a quick rate that ice
crystals do not have time to form. A second technique is to add a cryoprotectant before freezing. A
cryoprotectant is a chemical that interferes with ice formation. An everyday cryoprotectant is propylene
glycol or ethylene glycol, commonly used in car radiators as antifreeze. In our experiments, we will be
using glycerol as a cryoprotectant.
Selected References:
Scopes, R K (1994), Protein Purification , 3rd ed, Springer-Verlag, New York, pp 4-8 & 22-38.
Materials:
In cold room: bovine tissue, weigh boats, triple beam balance, sharp knife or scalpel, thermometer,
chilled 50 mM sodium phosphate, 10 mM EDTA, pH 7.5 buffer (use only the buffer in the cold room for
this lab), Waring blender, Teflon pestle, homogenizer and motor
In lab: ice bucket with ice, 250 mL beaker, 100 mL graduated cylinder, glass stirring rod, two 250 mL
centrifuge tubes, Beckman J2 preparative centrifuge and JA-14 rotor, 1.5 mL eppendorf tubes, pipetman
and pipet tips, eppendorf tube rack, glycerol, isopropanol/dry ice in dewar flask, -20°C freezer.
Safety:
The solutions and tissues used in this lab are not toxic and can be disposed of down the drain. Large
pieces of tissue will clog the drain so place them in the appropriate waste bucket. Wear safety glasses
and gloves at all times; use caution with knives/scalpels; hold homogenizer at sides, not bottom to avoid
injury; do not use centrifuge without assistance (even if you have experience with them); keep fingers
out of isopropanol/dry ice. Tie long hair to avoid it getting caught in the homogenizer. All broken
glassware and used Pasteur or graduated pipets must go in glass waste. Pipet tips must go in broken
glass or sharps waste containers.
Procedure:
1) Before you start working be sure to chill/prepare the appropriate materials
2) Each group of students will be assigned a tissue (liver, kidney or skeletal muscle) by the TA – you
may not choose your own.
3) In the cold room, weigh ~20 g of assigned tissue using a weigh boat and scale. Record the mass
measured.
4) In a beaker, mix the tissue with 80 mL of ice cold buffer (50 mM sodium phosphate, 10 mM
EDTA, pH 7.5). Chill on ice until <5°C.
LDH Exercise 1: Preparation of Isoenzymes of LDH from Tissue Extracts
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5) Transfer mixture to a blender. Blend on high speed for 30s. Check temp – keep below 20°C.
Repeat blending and checking temperature until the tissue forms a homogeneous suspension.
Return suspension to a pre-chilled beaker and chill on ice.
6) Label and place one 250 mL centrifuge tube in an ice bucket to cool during the next steps.
7) Stir the suspension well using a glass stirring rod. Measure the volume of the suspension, then
measure ~50 mL with a graduated cylinder. Record the volume then pour into homogenizer (do
not overfill, it will only hold about 50 mL) and record the volume used. Homogenize the
suspension using an electronic motor and Teflon pestle. Periodically check the temperature.
Stop and chill on ice if the temp becomes >20°C. The suspension should be completely
homogeneous with no chunks of tissue at the end of this step and the pestle should be able to
pass smoothly through the suspension to the bottom of the glass tube.
8) Clean the blender by washing with soap and a brush, then rinse with tap, DI, and distilled water.
Return the blender to the cold room.
9) Transfer the ~50 mL homogenate to a chilled 250 mL tubes and bring to the centrifuge in an ice
bucket. Balance the tubes by partnering with another group, remove sample from the heavier
tube to balance. In the interest of lab period time, please be sure to fill the centrifuge rotor with
tubes from other groups.
10) Centrifuge using a JA-14 rotor at 13,000 rpm at 4°C for 20 min. Allow the TA to operate and
demonstrate the use of the centrifuge.
11) While centrifuging:
a. Clean up the homogenizer and pestle as done with the blender and dispose of any
remaining suspension into the sink.
b. Clean up the area in the cold room around the blender and homogenizer. Any chunks of
tissue should go into the trash can in the cold room, not the sink.
c. Have your TA check your area in the cold room, including homogenizer, blender and
bench area before leaving.
d. Prepare ten 1.5 mL Eppendorf tubes by labeling each tube with: tissue type (Kidney,
Liver, or S. muscle), initials or last name and date using the alcohol resistant VWR
markers. Place tubes on ice in an ice bucket. Label one Eppendorf tube rack with your
name, “LDH Ex. 1”, date, and the tissue.
12) Pour the supernatant into a clean, chilled graduated cylinder and record the volume. Be careful
to avoid the pellet or any floating particles. You can transfer the liquid using a Pasteur pipet if
there is a lot of fat.
13) In one 15 mL Falcon tube (disposable plastic tube), add 9 mL of your supernatant and gently mix
with 1000 L of glycerol, mix until the clear layer of glycerol no longer persists. (Pre-chill the
falcon tube to keep your sample cold. Do not vortex).
a. Glycerol is highly viscous, pipette very slowly. You may also cut the end of the pipette tip
to ease with dispensing.
14) Dispense 1 mL aliquots into each Eppendorf tube prepared in step 11. Glycerol must be
completely mixed before dispensing.
15) Freeze in isopropanol/dry ice (as demonstrated by TA) and store in rack at -20°C.
LDH Exercise 1: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
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**The addition of glycerol to tissue extracts before freezing helps preserve enzymatic activity. Mix
thoroughly by inversion and/or pipetting to disperse the glycerol. Rapid freezing in dry ice/isopropanol
also helps preserve activity. Add glycerol to all aliquots intended for freezing in this and subsequent
exercises.
Planned Use of Tissue Homogenate Aliquots
Each aliquot = 1 mL homogenate + 0.1 mL glycerol
Exer 2: Spec Assay of LDH
Exer 4: electrophoresis
Exer 3: kinetics-kidney
Exer 3: kinetics-skel muscle
Backup
kidney*muscle*liver
1
1
1
2
2
2
8
------8
--4
4
2
*If Ex. 3 is not on the schedule, only five aliquots of muscle and kidney are needed.
Questions to think about:
1.
2.
3.
4.
5.
In which cell compartment is LDH found? How does this relate to the method used in releasing it
from the tissue?
Why are kidney and skeletal muscle chosen for the kinetic analysis (Exercise 3)?
Why do you keep everything that comes into contact with your tissue cold?
Why might a tissue such as the pancreas not be a good source to choose for the isolation of
LDH?
Why is EDTA included in the buffer?
LDH Exercise 1: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
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Exercise 2 Enzymatic Assay of LDH by Spectrophotometry
Key Principles:
Principles of enzyme kinetics, use of the spectrophotometer to quantify enzymatic activity; units for
enzymatic activity.
Background
The tissue extract that you prepared in the previous laboratory consists of a mixture of proteins. Cells
contain a large variety of proteins in the cytosol to carry out the chemical reactions necessary for life.
One of those proteins is lactate dehydrogenase (LDH). Now that you have the tissue homogenate, one
of the most fundamental questions to ask is, “How much LDH is in the extract?” How might you go
about quantifying the amount of LDH present in the crude extract?
Enzymologists typically use enzymatic reactions to determine the amount of enzyme present in a
solution. Reactions catalyzed by enzymes may be somewhat different than other chemical reactions
you may have come across. For many chemical reactions, the reaction occurs spontaneously if you place
the proper reactants together. On the other hand, reactions catalyzed by enzymes often do not occur at
an appreciable rate in the absence of the enzyme catalyst. Consider the case for table sugar, sucrose.
There is a significant amount of chemical energy present in sucrose and release of the energy can occur
in the presence of oxygen. However a large amount of energy (fire) must be input in order for the
release to occur (through combustion). In biological systems, the energy present in sucrose is released
slowly through a set of enzymes (glycolysis, Krebs cycle, and oxidative phosphorylation enzymes) to
allow the energy to be captured and put to use for biological processes. Because the reactions that
enzymes catalyze are often slow or nonexistent without the enzyme, reaction rates are directly
proportional to the amount of enzyme present. This should make intuitive sense. The more of the
enzyme present to catalyze the reaction, the more substrate (for enzymes, reactants are known as
substrate) will be converted to product, ultimately resulting in a greater overall reaction rate. The
amount of enzyme present in your LDH extract is related to how quickly your extract can convert the
substrate (pyruvate) into product (lactate).
There are a couple of important assumptions that should be pointed out, though. In order to
understand our first assumption, we need to determine how reaction rates are measured. Based upon
what you know about chemical reactions, as a reaction proceeds, reactants are consumed and products
are formed. Thus reaction rates can be measured by the rate of disappearance of reactant or the rate of
appearance of product. The first assumption we will make is that LDH is the only enzyme in your extract
that catalyzes the conversion of pyruvate to lactate. You should be able to understand how another
enzyme could interfere with the measurement if it also could convert pyruvate into lactate.
Nonetheless, it is reasonable to assume LDH is the only enzyme that will act on pyruvate since enzymes
tend to have a high specificity for their cognate substrates and it is unlikely that any of the other protein
in the extract will have an appreciable rate of reactivity with pyruvate.
The second assumption is that the reaction rate that we measure arises from the situation where all
enzyme molecules are bound to substrate virtually all the time. To understand why this is important,
consider the following argument. Say you wish to measure the amount of LDH enzyme present in your
extract and you are going to determine this by measuring the rate at which pyruvate is consumed. If
you saturate the system with the substrate, pyruvate, such that all the LDH is bound, then the measured
reaction rate will have resulted from the participation of all enzyme molecules in the extract. If you
LDH Exercise 2: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
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Page 25
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don’t add enough pyruvate, then only a fraction of the enzyme in the extract will be bound to the
substrate and at any point in time and you will underestimate the LDH content. Due to this argument, it
is important to flood the system with substrate in order to obtain an accurate assessment of the amount
of LDH present in your extract.
The question then becomes, “How much substrate is
enough?” This question leads directly into core
principles of enzyme kinetics which will be the focus
in the next experiment. However, you should
1/2Vmax
already be familiar with these concepts from your
prerequisite biochemistry coursework. If you recall,
KM, the Michaelis constant, is the concentration of
substrate at which the reaction velocity reaches one
KM
half the maximum reaction velocity. Therefore, at
[Substrate]
concentrations of substrate much greater than the
Figure 6 Plot of velocity vs. [Substrate]. The plot is govern
KM, all of the enzyme molecules become bound by
𝑽
𝑺𝒖𝒃𝒔𝒕𝒓𝒂𝒕𝒆
by the equation: 𝒗𝒆𝒍𝒐𝒄𝒊𝒕𝒚 = 𝒎𝒂𝒙
. The Michealis substrate and the reaction velocity reaches its
𝑲𝑴+ 𝑺𝒖𝒃𝒔𝒕𝒓𝒂𝒕𝒆
constant (KM) is the concentration of substrate at which the maximum (Vmax). Though you will not experimentally
reaction velocity is half maximum. When the concentration determine the KM value of LDH for pyruvate until
of substrate is much greater than KM, the velocity of the
Exercise 3, it is important to note that for this
reaction reaches its maxiumum (Vmax).
experiment, you will be measuring the Vmax since the
concentration of substrate that you will use has
already been assigned such that its concentration is much larger than the KM.
(Absorbance)
velocity
Vmax
Wavelength
+
+
Figure 7 Relationship between NAD and NADH. Left: Structures of NAD and NADH. Ribo=ribose,
+
ADP=adenosine diphosphate. Right: NAD and NADH absorbance spectra differ. NADH absorbs light
+
at 340 nm whereas NAD does not.
In this experiment, you will be conducting an enzymatic assay, or a test. This assay will be undertaken
primarily to gain experience with conducting an enzymatic assay, to identify whether LDH is present in
your extract, and to quantify the amount of LDH that was harvested from your tissue. Most enzymatic
assays are conducted by monitoring the rate of formation of product or the rate of disappearance of
substrate. Given that LDH catalyzes the reaction shown below, one can assay for LDH by monitoring the
rate of pyruvate or NADH consumption.
LDH Exercise 2: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
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CH3COCOO- + NADH + H
(pyruvate)
Page 26
Fall 2018
CH3CHOHCOO- + NAD+
(lactate)
For Exercise 2, you will take advantage of a special property of NADH to measure its consumption. The
structure of NADH changes upon oxidation to NAD+, and this conversion causes it to no longer be able to
absorb light at 340 nm wavelength. Therefore, as the LDH reaction proceeds, the reaction rate can be
monitored by observing the rate of decrease in absorbance at 340 nm.
To recap, you will be using an enzyme kinetic assay to determine the amount of LDH present in your
extract. The assay will consist of mixing the substrates, pyruvate and NADH, to the extract which
presumably contains active LDH. Accurate determination of the amount of LDH present is only obtained
when substrate concentrations are much larger than their KM values. For this experiment, the
concentrations of substrate recommended in the protocol will satisfy this requirement. Once the
components are mixed, NADH (which absorbs light at 340nm) is consumed as the reaction proceeds. The
rate of reaction is directly proportional to the amount of enzyme present in the sample and is often
referred to as the activity. More active samples have more enzyme. Therefore, you can think of activity
as a representing how many grams or moles of enzyme are present. If student A calculates that she has
5 activity units of enzyme activity in her extract whereas student B calculates 10 activity units for his,
then student B has harvested twice as much enzyme.
There are various ways of expressing a sample’s activity. One of the most common is units, which is
defined as the number of moles of product formed/min. A related measurement is activity
concentration which is exactly as its name implies (i.e. the concentration of the activity, reported in
activity/mL or moles of product formed/min/mL). The relationship between the rate of decrease in
absorbance and activity can be derived from the Beer-Lambert law and knowledge of the LDH reaction’s
stoichiometry.
𝐴=∗𝑐∗𝑙
Eq. 2-1
𝛥𝑐𝑁𝐴𝐷𝐻
=
∗
∗𝑙
𝑁𝐴𝐷𝐻
𝑡
𝛥𝐴𝑁𝐴𝐷𝐻
1 𝑡
𝛥𝑐𝑁𝐴𝐷𝐻
∗
=
= 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑡
𝑁𝐴𝐷𝐻 ∗ 𝑙
𝑡
Eq. 2-2
𝛥𝐴𝑁𝐴𝐷𝐻
Eq. 2-3
However, the activity concentration shown in the equations above describes the rate of substrate used
not product formed. Since the stoichiometric ratios of reactants to products all equal one, the rate that
substrate is consumed can be related to the rate that product is formed as follows:
𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑢𝑠𝑒𝑑 ∗ −1 = 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑓𝑜𝑟𝑚𝑒𝑑
Care must be taken when considering activity in terms of activity vs. activity concentration. If student A
calculates that she has 5 activity units/mL of enzyme activity in her extract whereas student B calculates
10 activity units/mL for his, has student B harvested twice as much enzyme? Answer: Not necessarily.
He could have, but there’s one important assumption you’re making if you said he has harvested twice
as much as student A. Do you know what that assumption is?
Another way of looking at it is to realize that activity is an extensive property whereas activity
concentration is an intensive property.
LDH Exercise 2: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
CHEM 108
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Selected References:
Everse, J., and Kaplan, N. O. (1973) “Lactate Dehydrogenase: Structure and Function.” Advances in
Enzymology, Vol 37 (Meister, A., ed.), John Wiley and Sons, New York, pp. 61−133.
Kyte, J. (1995) Mechanism in Protein Chemistry, Garland Publishing, New York, pp. 145−174. Scopes, R.
rd
K. (1994) Protein Purification, 3 ed, Springer−Verlag, New York, pp. 50−56, 62−70. Segal, I. H. (1976)
nd
Biochemical Calculations, 2 ed, Wiley, New York, pp. 208 −323.
Materials:
In lab: ice bucket with ice, tissue extract (prepared in Ex. 1 – thaw one tube, keep on ice and discard
after use), 20, 200 and 1000 µL Pipetman, vortexing mixer, Eppendorf tubes for dilutions, 13 x 100 mm
glass culture tubes, Parafilm, 100 mM Sodium Phosphate, pH 7.5 (RT - for dilutions), 500 mM Sodium
phosphate, pH 7.5 (RT - for assays), 15 mM NADH (in your sections -20°C – take one tube and discard
after use), 100 mM Sodium pyruvate (in cold room – take one tube and discard after use)
Safety: the solutions and tissues used in this lab are not toxic and can be disposed of down the drain.
Wear safety glasses and gloves at all times. Glass tubes should be disposed of in glass waste along with
graduated and Pasteur pipettes.
Procedure:
1) Thaw one tube of your tissue extract (warm with hands) and store on ice.
2) Make a 1:10 dilution – mix 50 µL of extract with 450 µL of 100 mM sodium phosphate, pH 7.5 in
an eppendorf tube. Mix well and store on ice (you may vortex, but avoid frothing). Be careful
when pipetting, the extract is thick and it is easy to transfer more than intended. Wipe the
outside of the tip with a KimWipe if you see any excess on the pipette tip. Residue found at the
end of a pipette tip after measuring is unavoidable, therefore one should always refrain from
pipetting small volumes – less than 5 µL - as it introduces unnecessary error.
3) Make a 1:100 dilution – mix 5 µL of undiluted extract with 495 µL of 100 mM sodium
phosphate, pH 7.5 in an eppendorf tube, mix well with vortex and store on ice. Extract at this
dilution may lose activity over time. Do not make it until it is needed, and remember to store
the diluted extract on ice.
4) Prepare the Spec 20 (Appendix in lab manual)
a. Check wavelength and filter settings
b. Set 0 (no cuvette) and 100% (water) transmittance
c. Switch setting to absorbance
5) Prepare assay mix. You can make several of these in advance.
6) Mix the following in a glass culture tube (do not mix the enzyme until just before assaying; also
avoid touching the lower portion of the glass tube with your hands since debris will scatter light
and create error in the Spec 20 absorbance reading; similarly, do not store the assay tubes on
ice since water will condense on cold tubes which will interfere with the Spec 20 readings)
a. 0.600 mL 500 mM sodium phosphate, pH 7.5 (RT)
b. 0.030 mL 15 mM NADH (on ice)
c. 0.030 mL 100 mM sodium pyruvate (on ice)
d. 2.29 mL of water
LDH Exercise 2: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
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Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
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i. Tip: If you measure 2.50 mL using the P1000, it is inconvenient to pipette 1000
L twice, change the pipette, then measure 500 L. The fastest way: with a
P1000, change the setting to 0.833 mL and pipette 3 times.
7) Cover with parafilm and invert to mix
8) Put in spectrophotometer and check to see that A340 is approximately 0.5 – 0.8. If not, remake
mix. If it still isn’t, add more or less NADH to adjust.
Assay the extract
9) Add 50 µL of diluted tissue extract, either 1:10 or the 1:100 (Note: kidney and liver may have lower
activity concentrations), cover with parafilm and invert to mix.
10) Wipe the tube to be sure it is clear of smudges and condensation.
11) Insert in Spec 20, record initial A340 reading and at 30 s intervals for 3 min.
12) The decrease in A340 needs to change linearly over the course of the 3 min. This usually
happens when the decrease is 0.1 – 0.2 OD units/min over the course of 3 min. No need
to wait the full 3 min if the decrease is not within this range, or if it is not linear.
a. If the decrease is too small, then the noise of the spectrophotometer may significantly
interfere with the experiment. A reaction that is too slow results from not adding
enough enzyme. Make a more concentrated enzyme dilution and try again.
b. If it is too fast, then the reaction will slow as substrate gets used and becomes scarce.
Reactions that are too fast are caused by adding too much enzyme. Try diluting the
enzyme and try again.
13) Once you have correct dilution, repeat twice to get 3 good assays.
14) Cleanup: discard all solutions in drain, turn off Spec20.
15) Have your TA make a table on the chalkboard and fill in your info
Group (names)
Tissue
Dilution of Extract
Volume of Diluted
Extract added to Assay
LDH Exercise 2: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 29
Fall 2018
0.9
0.8
0.7
y = -0.0034x + 0.7996
R² = 0.9983
A340
0.6
0.5
0.4
0.3
0.2
0.1
0
0
20
40
60
80
100
120
140
160
180
200
time (s)
Figure 8 Analysis of LDH enzyme activity using spectrophotometry
Handling the Data
For this experiment, you can calculate the amount of enzyme that was present in the tissue extract as
enzyme activity. From the background for this exercise, the amount of enzyme is related to activity
concentration which can be calculated from the change in A 340 over time. Presumably you will have
collected three datasets, each consisting of measurements of decreasing A 340 over a 3 min time course.
First, plot the data as A340 vs. time (see Figure 8). Calculate a linear regression and determine the slope.
𝛥𝐴
The slope will equal 340. Then you can use the Beer-Lambert Law to determine activity concentration.
𝑡𝑖𝑚𝑒
𝛥𝑐𝑁𝐴𝐷𝐻
= 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝛥𝐴340
1 =
∗
𝑡
𝑡
340 ∗ 𝑙
340 for NADH = 6220 M-1 cm-1
l for the assay tube = 1 cm
Eq. 2-4
Activity concentration is typically expressed with the units mole (product formed)/min/mL. Be sure to
perform any appropriate unit conversions. This might be something similar to what follows:
𝛥𝐴340
𝑡
1 𝑚𝑜𝑙𝑒
1𝐿
1𝐸6µ𝑚𝑜𝑙𝑒 𝛥𝑐𝑁𝐴𝐷𝐻
𝐿 ∗
∗
∗
∗
∗
=
= 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
340 ∗ 𝑙 𝑚𝑖𝑛
1𝑀
1000𝑚𝐿
𝑚𝑜𝑙𝑒
𝑡
1
60𝑠𝑒𝑐
Eq. 2-5
Take a moment to consider what this calculation represents. The change in absorbance occurred in the
3 mL assay so the activity concentration calculated represents the amount of enzyme in the assay. What
we want is a measure of how much enzyme is present in the tissue extract. There are essentially two
dilutions that were made from the tissue extract to the assay. The first is the dilution that you finally
settled upon in step 13). The second is the dilution made when mixing the assay. The diluted extract
was further diluted when you mixed 50 L of diluted extract to 3 mL of assay (a 1/60 dilution). To
determine the activity concentration of the tissue extract, multiply by the two dilution factors. Also be
sure to report an average for the 3 measurements.
LDH Exercise 2: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 30
Fall 2018
At this point, you will have calculated the activity concentration for your extract. You should be able to
calculate the total activity of LDH enzyme you purified and the amount of activity you purified per gram
of tissue. Be careful with the latter calculation, however. Review your protocol and consider how each
of the steps might affect the calculation.
Questions to think about:
Reduced nicotinamide adenine dinucleotide has an absorption maximum at 260 nm as well as at 340
nm. Why do you measure LDH activity at 340 nm rather than at 260 nm?
Solutions of NADH and pyruvate must be made fresh each day. Why?
LDH Exercise 2: Preparation of Isoenzymes of LDH from Tissue Extracts
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 31
Fall 2018
Exercise 3 Kinetic Analysis of LDH
Key principles:
Determination of kinetic constants (KM, KI, and Vmax), competitive inhibition, noncompetitive inhibition,
uncompetitive inhibition
Background
Characterization of an enzyme often consists of determining its kinetic constants, K M and Vmax. The
importance of these constants were mentioned in the previous Experiment’s Background section. Vmax
is the maximum velocity that is reached when all of the enzyme is bound to its substrate. Under this
condition, the velocity serves as a measure of how much enzyme is present so it provides a means of
quantifying the amount of enzyme present in a solution, such as a tissue extract. The Michaelis
constant, KM, is the substrate concentration at which half the enzyme is bound to substrate. It provides
a guide for how good an enzyme is. The lower the KM, the less substrate is needed to fill the enzyme
active site and the better the enzyme is. Along the same line of reasoning, the K M is a gauge for how
much substrate needs to be added to the assay in order to reach Vmax.
Referencing Figure 6 should assist with understanding these concepts. Figure 6 also shows that velocity
measurements taken at multiple substrate concentrations will yield a curve in which V max is the
asymptote and KM is the substrate concentration where the velocity is ½*Vmax. Since you know how to
measure velocities from the LDH enzymatic assay performed in Exercise 2, you technically have the tools
to measure the kinetic constants. The one problem you would face is defining the asymptote. Without
performing advanced regression analysis, you are left with “eyeballing” the location of the asymptote.
Since the KM calculation is dependent on the V max measurement, it will also be subject to error.
Rather than guessing where the asymptote is located, we will be using some mathematical tricks to help
us pinpoint the values of KM and Vmax. Let’s review the derivation of the Michaelis-Menten equation and
then we will examine the math manipulation that helps define KM and Vmax.
The general enzyme mechanism is defined as follows:
k1
k2
E+S
ES
E+P
k-1
E = enzyme
S = substrate
ES = enzyme/substarte complex
P = product
In this reaction, the conversion of ES to E and P is the rate limiting step, so the rate of the reaction is
defined as writtein in Eq. 3-1. It should be readily understood that the total concentration of enzyme in
the system, [ETotal], should equal the concentration of free enzyme and the concentration of
enzyme/substrate complex, as presented in Eq. 3-2. These two equations are the starting point for the
derivation of the Michaelis-Menten equation.
LDH Exercise 3: Kinetic Analysis of LDH
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Divide Eq. 3-1 by Eq. 3-2
Multiply each side of equation
the by [ETotal]
Define K as 𝐾 =
𝑀
M
𝐸 𝑆
𝐸𝑆
𝐸
Factor out 𝐾 from the
𝑀
numerator and denominator
𝐸
Cancelling 𝐾 results in the
𝑀
Michaelis-Menten equation
Take the reciprocal of both
sides of the equation (i.e. raise
both sides to the power of -1)
Separate the numerator terms
over the common denominator
Cancel [S] in the second term,
resulting in the LineweaverBurk equation
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𝑣 = 𝑘2 𝐸𝑆
Eq. 3-1
𝐸𝑡𝑜𝑡𝑎𝑙 = 𝐸 + 𝐸𝑆
Eq. 3-2
𝑣
𝑘2 𝐸𝑆
𝐸𝑡𝑜𝑡𝑎𝑙 = 𝐸 + 𝐸𝑆
𝐸𝑡𝑜𝑡𝑎𝑙 𝑘2 𝐸𝑆
𝑣=
𝐸 + 𝐸𝑆
𝑉𝑚𝑎𝑥 𝐸𝑆
𝑣 = 𝐸 + 𝐸𝑆
𝐸 𝑆
𝐾
𝑉
𝑣 = 𝑚𝑎𝑥 𝑀
𝐸 𝑆
𝐸 + 𝐾
𝑀
𝐸
𝑉𝑚𝑎𝑥 𝑆
𝐾
𝑣= 𝑀
𝐸
(𝐾𝑀 + 𝑆 )
𝐾𝑀
Eq. 3-3
𝑉𝑚𝑎𝑥 𝑆
𝑣= 𝐾 + 𝑆
𝑀
Eq. 3-8
1 𝐾𝑀 + 𝑆
𝑣 = 𝑉𝑚𝑎𝑥 𝑆
Eq. 3-9
1
𝐾𝑀
𝑆
𝑣 = 𝑉𝑚𝑎𝑥 𝑆 + 𝑉𝑚𝑎𝑥 𝑆
Eq. 3-10
1
𝐾𝑀 1
1
𝑣 = 𝑉𝑚𝑎𝑥 𝑆 +𝑉𝑚𝑎𝑥
Eq. 3-11
Eq. 3-4
Eq. 3-5
Eq. 3-6
Eq. 3-7
That math “trick” that will help us define the kinetic constants is taking the reciprocal of both sides of
the Michaelis-Menten equation to yield Eq. 3-9. Eq. 3-11 may look complicated in the end, but it is
actually the equation for a line. The general equation for a line is y = mx + b, thus in the LineweaverBurk plot y = 1, m = 𝐾𝑀 , x = 1 , b = 1 . Creating a plot of 1vs. 1 will reveal K and V as shown in
𝑣
𝑉𝑚𝑎𝑥
𝑆
𝑉𝑚𝑎𝑥
𝑣
𝑆
M
max
Figure 9.
5
1
𝑉𝑚𝑎𝑥
3
5
1/v
0 .0 2
5
1
5
0
0
5
1
-𝐾
𝑀
1/[S]
Figure 9 Lineweaver-Burk plot. The x- and y-intercepts
identify K and Vmax.
This experiment is very similar to Experiment 2. You
will be using the same LDH assay as before to
determine the kinetic constants of LDH. In order to
complete a Lineweaver-Burk plot, you will need to
perform measurements of velocity at different
substrate concentrations. One key difference you
should note is that the concentration of substrate will
be far lower than what was used for the previous
exercise. This is done since we are not interested in
obtaining a measurement of the amount of protein
for this exercise. For determining the kinetic
constants, the substrate concentrations used for the
assay should be near the KM since the greatest
differences in velocity are be observed at these
concentrations (see Figure 6).
LDH Exercise 3: Kinetic Analysis of LDH
Techniques in Protein Chemistry
CHEM 108
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Enzyme Inhibition
You will also be characterizing the effects of oxamic acid on LDH. Oxamic acid interacts with LDH and
slows the reaction. Thus it is an inhibitor of LDH. There are several manners in which inhibitors bind to
and inhibit enzymes. The three main methods are known as competitive, noncompetitive, and
uncompetitive inhibition.
Figure 10 Competitive, noncompetitive, and uncompetitive inhibition. The reaction mechanisms, Lineweaver-Burk equations,
and Lineweaver-Burk graphs are shown. I = inhibitor
For a competitive inhibitor, the inhibitor molecule and the substrate bind the enzyme mutually
exclusively. Usually this inhibitor binds at the enzyme active site and thereby “competes” with the
substrate. In this type of inhibition, the Vmax remains constant regardless of the presence of inhibitor.
KM, on the other hand appears to become larger at increasing inhibitor concentrations. In contrast, a
noncompetitive inhibitor can bind to and inhibit the enzyme regardless of whether substrate is bound.
In this case, the KM remains constant in the presence of inhibitor, but V max appears to decrease as
inhibitor concentration increases. An uncompetitive inhibitor only binds to the enzyme when the
substrate is already bound. Both KM and Vmax appear to change when inhibitor is added for an
uncompetitive inhibitor.
The Lineweaver-Burk equations for each of the inhibitor types can be derived analogously to the
derivation provided in Eq. 3-1 through Eq. 3-11. The inhibition constant, KI, is defined as the ratio of the
concentration of the reactants multiplied together vs. the concentration of the products. Through the
derivation for each type of inhibitor, it can be shown that KMapp and Vmaxapp can be defined as the
following expressions.
Mathematical expressions for KI and the derivation for the equations below are provided in the
Appendix.
LDH Exercise 3: Kinetic Analysis of LDH
Techniques in Protein Chemistry
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Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Competitive Inhibition
Noncompetitive Inhibition
Uncompetitive Inhibition
Uncompetitive Inhibition
𝐾
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Fall 2018
𝐼
= 𝐾 (1 +
𝑀𝑎𝑝𝑝
𝑀
𝑉𝑚𝑎𝑥 𝑎𝑝𝑝=
𝐾𝑀𝑎𝑝𝑝 =
𝑉𝑚𝑎𝑥𝑎𝑝𝑝
𝑉𝑚𝑎𝑥
)
𝐾𝐼
𝐼
Eq. 3-12
Eq. 3-13
1+𝐾
𝐾𝑀𝐼
(1 +
𝐼
)
𝐾𝐼
𝑉𝑚𝑎𝑥
=
𝐼
(1 + )
𝐾𝐼
Eq. 3-14
Eq. 3-15
Important Notes on the Procedure:
This experiment will take you a long time if you do not come into lab prepared (i.e. without having a
good idea of what you are going to do). There are a lot of enzymatic activity assays that need to be
measured. Each Lineweaver-Burk plot requires many assay measurements taken at different substrate
concentrations. The entire experiment needs to be repeated in the presence of inhibitor to determine
the inhibition type and KI. The good thing is that there are a lot of students in each section so we are
going to divide the work up among the groups, then share the data at the end. Your classmates are
counting on you to do your part so that the class results will be interpretable!
Data from the entire section will be combined for the formal report – be sure that you get data from
your TA and other groups for the different extracts.
Materials:
In lab: ice bucket, P20, P200 and P1000 µL Pipetman and tips, 5 mL graduated pipets and pipet bulb,
vortexing mixer, Eppendorf tubes for dilutions, 13 x 100 mm glass culture tubes, Parafilm, timer, 100
mM Sodium Phosphate, pH 7.5 (RT - for dilutions), 500 mM Sodium phosphate, pH 7.5 (RT - for assays)
In freezer: tissue extract (prepared in Ex. 2 – thaw just one at a time), 15 mM NADH (in your section’s 20°C freezer – take one tube and discard after use)
In cold room: 100 mM Sodium pyruvate (1 tube/group), 20 mM Oxamic acid,
Safety: The solutions and tissues used in this lab are not toxic and can be disposed of down the drain.
Wear safety glasses and gloves at all times. Glass tubes should be disposed of in glass waste along with
graduated and Pasteur pipettes.
Time guideline:
(10 min) Create a pooled extract for the class
(30 min) Class determination of proper dilution/setup pyruvate dilutions (tasks divided between group
members
(30 min) Add reagents to tubes in preparation for assay
(3 min*8 assays = 30 min) Assay 8 pyruvate concentrations without inhibitor (3 min*8 assays = 30 min)
(3 min*8 assays = 30 min) Assay 8 pyruvate concentrations with inhibitor
(3 min*8 assays = 30 min) Duplicate: Assay 8 pyruvate concentrations without inhibitor
(3 min*8 assays = 30 min) Duplicate: Assay 8 pyruvate concentrations with inhibitor
Procedure:
Day 1
1) We will be using muscle tissue extract for this experiment. Identify groups that used muscle
tissue extract.
LDH Exercise 3: Kinetic Analysis of LDH
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 35
Fall 2018
2) Of those groups, identify the groups that had high LDH activity (extracts that were made more
dilute should have more enzyme activity). Refer to the table that you filled out in the previous
exercise.
3) Have two to three groups with the most active extracts donate one or two 1 mL aliquots for the
class experiment. The class should try to start with ~4 mL of pooled extract.
4) Have one member of each group optimize the dilution of pooled extract needed to observe a
decrease of A340 at a rate of 0.1-0.2/min in the LDH assay outlined in Exercise 2. The other
member should work on making the pyruvate dilutions (step 5).
a. The class should not get tied up with this measurement. You have a clue as to what
dilutions to try, the groups that donated the enzyme told you what worked before.
b. Remember to dilute the extract in 100 mM sodium phosphate, pH 7.5
c. Write your group name, the dilution, volume of diluted extract used for the assay, and
the estimated dA/dt on the board.
d. Once 3 dilutions are reported that agree with each other (or are nearly equal), the
entire class should use that dilution for the rest of the experiment.
5) Prepare pyruvate at concentrations that are 10, 1, 0.8, 0.6, 0.5, 0.4, 0.3, and 0.2 mM based
upon the following table. Keep in mind that the final concentrations will be 10-fold less since
you will add 0.3 mL of the pyruvate solutions to a 3 mL assay. KEEPING THIS IN MIND WILL BE
IMPORTANT FOR YOUR CALCULATIONS!
Pyruvate Dilution Table
Desired
[pyruvate]
Volume of
100 mM
pyruvate
Volume
water
Final volume
10 mM
1 mM
0.3 mL of
100 mM
0.03 mL of
100 mM
2.7 mL
2.97 mL
3 mL
3 mL
0.8 mM
0.6 mM
0.5 mM
0.4 mM
0.3 mM
0.2 mM
3 mL
3 mL
3 mL
3 mL
3 mL
3 mL
6) Prepare the Spec 20 as done in LDH Exercise 2.
7) Assay LDH activity at all of the pyruvate concentrations. At this point, measure the rate of
decrease in A340 over the course of 3 min at 30 sec intervals one time. Do not be alarmed if the
rate of decrease does not fall within the 0.1-0.2/min range. The reaction rate is expected to
decrease as the concentration of pyruvate decreases (do you know why???) Use the dilution
and volume of extract agreed upon by the class.
Use the following recipe for the reaction:
0.6 mL of 500 mM sodium phosphate buffer, pH 7.5
0.030 mL of 15 mM NADH
0.30 mL of pyruvate dilution, careful this volume is different than in Exercise 2
XX mL of diluted extract (as what is decided in class); probably 0.050 mL
XX mL of water (to make the total volume 3 mL)
Total: 3 mL
Day 2
8) Assay LDH activity at the same pyruvate concentrations in the presence of the oxamic acid
inhibitor. Your TA will assign you with a concentration of oxamic acid (20, 50, or 100 M oxamic
acid). At this point, measure the rate of decrease in A340 over the course of 3 min at 30 sec
intervals for each pyruvate concentration one time. This way you will have one complete data
set. If there is time, repeat the measurements with and without inhibitor to obtain duplicate
LDH Exercise 3: Kinetic Analysis of LDH
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
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data sets (i.e. repeat steps 7) and 8)). The rate of decrease at each pyruvate concentration is
expected to be less than without inhibitor.
Use the following recipe for the reaction:
0.6 mL of 0.500 M sodium phosphate buffer, pH 7.5
0.030 mL of 15 mm NADH
0.30 mL of pyruvate dilution
XX mL of XX oxamic acid (check your stock concentration, you may need to dilute)
XX mL of diluted extract (the same as what was used in step 7)
XX mL of water (to make the total volume 3 mL)
Total: 3 mL
9) It should take ~30 min to complete all of the dA/dt measurements for all of the pyruvate
concentrations. Inform your TA if there is not enough time to complete one or both duplicate
sets. No penalty if you genuinely ran out of time. Minus points for those who leave early
despite having time to complete the trials.
10) Analysis requires pooling data from the entire section. You must provide your group’s data by
the end of the lab period (or within 2 hrs following lab). Because the data will be combined, the
data must be accessible to all before you leave. Your TA will provide details.
LDH Exercise 3: Kinetic Analysis of LDH
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
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Graph the data
Graph Abs vs. time for each trial and calculate enzyme activity (µmol/min) for each trial (plus and minus
inhibitor). It may be helpful to set up an EXCEL file with the necessary calculations to save time. You
should note that the calculation for velocity is slightly different for this experiment as compared with
Exercise 2. We are no longer interested in the total activity of the extract. The activity of the total
extract can be measured if the assay contains an excess of substrate so that Vmax is measured. In this
experiment, we are using mainly lower substrate concentrations. For this exercise, we are interested in
how the velocity changes with respect to substrate concentrations (you’re going to plot v vs. [S]). So in
which tube do you know both the velocity and the substrate concentration? You need to calculate the
substrate concentrations and the corresponding velocities for this tube.
Graph v0 vs. [pyruvate] for all trials.
v0 is the average enzyme activity (µmol/min) of the two trials
Graph 1/v0 vs. 1/[pyruvate]. This is the Lineweaver Burk Plot.
Each student should perform the kinetic analysis using the data from the class to determine K M, Vmax,
and KI for the inhibitor. You may use the following information to calculate K M, Vmax and KI
To determine KM and Vmax in the absence of inhibitor from the Lineweaver Burk plot, use
slope = KM/Vmax
x-intercept = -1/KM
y-intercept = 1/Vmax
For a competitive inhibitor, Vmax does not change. KMapp, (where app designates the apparent kinetic
constant when in the presence of inhibitor) is determined from the x-intercept on the Lineweaver-Burk
plot in the presence of inhibitor).
𝐼
𝐾𝑀 = 𝐾𝑀 (1 + )
𝑎𝑝𝑝
𝐾𝐼
Since [I] is known, KI can be determined. You can calculate this at multiple inhibitor concentrations and
determine an average if multiple inhibitor concentrations had been used.
For noncompetitive inhibition, KM is unchanged and Vmax is decreased to Vmaxapp. Vmaxapp is found from
the y-intercept of the Lineweaver-Burk plot in the presence of inhibitor and can be used to calculate K I.
𝑉𝑚𝑎𝑥
𝑉𝑚𝑎𝑥𝑎𝑝𝑝 =
𝐼
(1 +
𝐾𝐼 )
For an uncompetitive
inhibition, both KM and Vmax are changed.
𝐾𝑀
𝐾𝑀𝑎𝑝𝑝 =
𝐼
(1 + )
𝐾𝐼
𝑉𝑚𝑎𝑥
𝑉
=
𝑚𝑎𝑥𝑎𝑝𝑝
𝐼
(1 +
𝐾 )
𝐼
Assistance for your lab report
LDH Exercise 3: Kinetic Analysis of LDH
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Results. All graphs should have a legend that is numbered (e.g. Figure 1), has a title, labeled axes, and a
descriptive legend. Sample calculations should be shown in an appendix/supplemental data section
placed at the end of the report.
Calculate an average enzyme velocity in µmol/min/mL for each conc of pyruvate in the presence and
absence of inhibitor using your data. This can be done using EXCEL using the slope function or the
average rate of change/min calculated from the data. A graph does not need to be made for each trial,
however you may want to create them and supply them as supplemental material. Include a table of all
v0 values for all pyruvate concentrations with i) no inhibitor, ii) with inhibitor in an
appendix/supplemental data.
Graph: v0 vs. [pyruvate] with inhibitor and without inhibitor on the same graph. Use the average values
for v0. Keep in mind that [pyruvate] is the final concentration of pyruvate in the assay, not the initial
dilutions that you made. Be careful with regression analyses. Which general equation should your plot
fit?
Graph 1/v0 vs. 1/[pyruvate] in the absence and presence of inhibitor. Use this graph to calculate KM and
Vmax for pyruvate and the average KI using the calculations given above.
Make a table giving the kinetic constants – KM, Vmax and KI .
Formal Report Discussion ideas
Discuss the meaning of the KM and Vmax values obtained for pyruvate in the tissue studied. Would you
expect a different value for lactate? Why or why not?
Compare the values obtained to published values.
Use the graphs to determine if the inhibitor is a competitive, noncompetitive, or uncompetitive inhibitor
and discuss how this was determined.
Look in the literature and determine the type of inhibitor it is expected to be.
Compare this to your results and discuss any differences.
Is this type of inhibition expected based on the structure of the inhibitor and the tissue type?
Compare the KI value you obtained to the literature value.
References
The references in the lab manual – particularly Evers & Kaplan, Advances in Enzymology and Holbrook,
The Enzymes are useful for constants and background on LDH. They are both long and not available
online so take some time and scan through them to find the needed information. Some additional
suggested references for LDH inhibition and kinetic constants
J. S. Nisselbaum, D. E. Packer, O. Bodansky (1964) Comparison of the actions of human brain, liver and
heart lactic dehydrogenase variants on nucleotide analogues and on substrate analogues in the absence
and in the presence of oxalate and oxamate, J. Biol Chem 239: 2830-2834.
D. T. Plummer and J. H. Wilkinson (1963) Organ Specificity and Lactate Dehydrogenase Activity Biochem
. J 87: 423 – 429.
W. B. Novoa, A. D. Winer, A. J. Glaid, and G. Schwert (1959) Lactate Dehydrogenase V. Inhibition by
oxamate and by oxalate J. Biol. Chem (1959) 234: 1143-1148.
Oxamate and pyruvate structures: http://biochemistry.wur.nl/pbc/ldh/structures.html
LDH Exercise 3: Kinetic Analysis of LDH
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Exercise 4 Separation and Visualization of LDH Isoenzymes by Electrophoresis
on Cellulose Acetate
Key Principles:
Isoenzymes; Tissue differences; Separation of nearly identical proteins using an electric field; Stain for
localization of activity of LDH; Inescapable reversibility of enzymatic reactions.
Description of Procedure:
You will be conducting electrophoresis for this exercise. Electrophoresis is simply movement of charged
particles through an electric field. In the experiment, a sample is placed in between two electrodes
(positive and negative) and a current is applied. The technique is typically used as a means to separate
the particles in the sample due to their physical properties. The charge to mass ratio is one factor that
affects the rate of migration of charged particles in an electric field. Particles that are more highly
charged migrate through the electric field faster than particles that are less charged. Recall from your
biochemistry class that proteins are composed of 20 amino acids. Each of the 20 amino acids is
distinguishable by its side chain. Some of the side chains contain charged functional groups. Lysine,
arginine, and (to a small extent) histidine are typically positively charged (they are more basic) at
physiological pH. Aspartic acid and glutamic acid are negatively charged (they are more acidic) at
physiological pH. Mass is another criteria that affects how particles move in an electric field. The more
mass a particle has, the more electric force is required to make it move. Thus migration rates are
inversely proportional to a particle’s mass in an electric field.
In theory, electrophoresis can be conducted in water or a buffer, however electrophoresis of proteins is
typically conducted in conjunction with a matrix. The matrix is a material that serves as a substance that
the proteins can interact with. One purpose of the matrix is to limit the diffusion of the proteins.
Without the matrix, any observed movement of the proteins could be attributed to diffusion as well as
the electric field. For this experiment, you will be using strips of cellulose acetate paper as the matrix.
The various LDH isoenzymes can be separated by electrophoresis because they have distinct isoeletric
points (pI), which is the pH at which the net charge on the molecule is zero. The pI of a protein is
determined by its amino acid composition. As mentioned above, amino acids having an ionizable
sidechain make the greatest contribution. Aspartate and glutamate have acidic sidechains, while lysine,
arginine, and histidine have basic sidechains (histidine is positively charged at pH <6). If a protein is in a
solution that has a pH equal to the protein’s pI, the net charge on the protein is zero. If the pH is below
the pI, the protein will have positive charge. If the pH is above the pI, the protein will have negative
charge. Thus the pI of a protein will affect the direction and rate of migration electrophoresis
experiments. The mass of each of the LDH isozymes is roughly equal so this this physical property does
not play much of a role in the migration rate for LDH. Note: although the goal of this experiment is
similar to that of an isoelectric focusing electrophoresis, this is a different technique. In the experiment
you will be performing, the pH is constant throughout the buffer chamber. A pH gradient is set-up for
isoelectric focusing.
Now that we have reached the genomics era of science, the amino acid sequences of many proteins
(including bovine LDH) are readily available. Instructions for retrieving the sequences for the bovine
LDH isoforms can be found on page 19. The amino acid sequence can be used as an input into a
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program which will calculate a theoretical pI. A popular program is ProtParam which is one of many
program tools presented on the ExPASy website. Using this program, you should be able to determine
the theoretical pI values of the heart and muscle isoforms and the 5 different isozymes they can form.
Based upon the prediction, you should be able to predict how the isozymes will migrate with respect to
one another.
Samples of tissue extracts from Exercise 1 will be applied to the center of cellulose acetate strips.
Voltage will be applied across the strips in order to separate the proteins in each sample. Remember
that these are tissue extracts and there are many proteins in the extract. Furthermore, most proteins
are not visible by eye. In order to observe only LDH, we will utilize an activity stain which takes
advantage of LDH’s enzymatic activity in order to reveal its location on the strip. The activity stain is a
mixture of reagents that produces a color on the strip at the location of the enzyme. The stain you will
be using is a mixture of NAD+ and the dyes nitroblue tetrazolium and phenazine methosulfate along with
lithium lactate. During the staining reaction, the enzyme catalyzes the reverse reaction compared with
that used for the activity assay in Exercise 2 (i.e. the reaction will consist of converting lactate into
pyruvate instead of pyruvate into lactate). In the presence of excess lactate and NAD+, NADH is formed.
The NADH reduces the phenazine methosulfate which in turn reduces the nitroblue tetrazolium, forming
a blue precipitate. This reaction takes place wherever LDH is present, so a blue precipitate locates LDH
on the strip. The number and intensity of the blue bands will be informative of the number of
isoenzymes and the relative amounts that were present in each tissue sample.
Selected References:
Berg, JM, Tymoczko, JL and Stryer, L (2007) Isoelectric focusing. In: Biochemistry, 6th ed, pp 73- 74.
Boyer, R (2006) Isoeletric focusing of proteins. In: Biochemistry Laboratory, Modern Theory &
Techniques. Pearson/Benjamin Cummings. pp 193-196.
Kyte, J. (1995) Structure in Protein Chemistry, Garland Publishing, New York, pp. 25-34. Malmstrom, J
(2006) Optimized peptide separation and identification for mass
Spectrometry-based proteomics via free-flow electrophoresis. J Proteome Res 5:2241-49.
Nelson, DL and Cox, MM (2008) Isoelectric focusing. In: Lehninger, Principles of Biochemistry, 5th ed, pp
90-91.
Scopes, R. K. (1994) Protein Purification, 3rd ed, Springer-Verlag, New York, pp. 250-258, 293306.
Materials:
In cold room: pure LDH from skeletal muscle and heart tissue (TA may have set out dilutions – check
labels); 100 mM pyruvate, Barbital buffer, pH 8.8 (handle with care); Electrophoresis Chambers with
strip holders; 50 mg/mL NAD+; 30% (w/v) sodium lactate; 3 mg/mL phenazine methosulfate; 3 mg/mL
nitroblue tetrazolium
In -20°C freezer: crude tissue extracts; 15 mM NADH,
In lab: 100 mM phosphate buffer; pH 7.5; 500 mM phosphate buffer, pH 7.5; Spec 20; pipets, tips and
eppendorf tubes; glass culture tubes; cellulose acetate strips; Tupperware and glass containers;
Whatman paper strips; plastic tray for staining; lightbox, scanner and camera; rulers
Safety: Barbital buffer is hazardous - wear gloves and safety glasses or goggles when handling - and
must go in the appropriate waste container not down drain. Phenazine methosulfate and nitroblue
tetrazolium may cause skin and eye irritation and must go in the appropriate waste container. (All three
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compounds may go in the same waste container – check labels) Use caution with the electrophoresis
chamber to avoid electric shock. Turn off power supply and disconnect from the electrophoresis
chamber at least 20s before removing lid and touching samples.
Time management guideline:
(60 min) Determine activity concentration of H4 and M4 and calculate how to make dilutions
(10 min) Locate extracts of other tissue samples from other groups and their reported activity
concentrations
(20 min) Make the sample dilutions
(10 min) Prepare electrophoresis
(90-120 min) Electrophoresis
(20 min) Activity stain
(20 min) Take picture of results
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Procedure:
1) Come to lab knowing the activity concentration of the LDH samples in the freezer that you
prepared in Exercise 1.
2) Assign one member of the group to sample preparation (starts on step 3) and the other to
prepare the electrophoresis chamber (starts on step 4). There will not be time to work together
on both steps. Take good notes and share them with all members of your group.
3) Sample preparation – As with most electrophoresis experiments, you need to determine how
much sample to add. If you add too little, you will not observe anything on your strips. If you
add too much, all of the isozyme bands will have so much activity that they will merge together
and you won’t be able to distinguish them. Sample preparation is critical and tends to take
students a long time. Work efficiently, yet carefully.
a. Standards – do this first!
i. You will be provided with a tube of purified skeletal muscle (M4) or heart (H4)
LDH. These standards are supplied by a company and their activities have
already been determined (reported as U/mL where 1 U = 1 Unit = 1 mole
product formed/min). However, they may have been on the shelf for a while so
we need to determine the activity concentration at the time of this experiment.
**TAs: make 500 L of a 1/10 dilution of the H4 and M4 standards. Remember
which tube you used as you will use these throughout the class. Assign half the
class to H4 and the other half M4. Distribute 30 L (save the excess on ice).
This is the “diluted stock”.
ii. Each group will probably need to further dilute the “diluted stock” by ~1/100.
Report the activity concentration for the “diluted stock” on the board. Once
there are 3 activity concentration measurements that are in agreement for each
standard, take the average for H4 and the average for M4 and have the class
use those values.
b. You will need kidney, liver, and skeletal muscle extracts for this experiment. Find groups
that possess extracts you are missing and ask them for their activity concentrations.
Kidney and liver may have low activity concentrations.
Try to find a sample that has >15 µmol/min/mL. If this is not possible, use the highest
and either pretend it is 15 µmol/min/mL or else adjust the volume you load onto the
strips accordingly.
c. Calculate the dilution needed to make all of your samples (M4, H4, kidney, liver, and
skeletal muscle) at a concentration of 15 µmol/min/mL, and prepare samples at this
dilution. Consider checking with your TA to see if your dilution is accurate. This step is
important so that all of your strips get the same amount of enzyme.
4) Preparing for Electrophoresis – done in the cold room, except for labeling the strips.
a. Working with gloves, obtain 5 cellulose acetate strips. The blue strips are spacers – do not
use them.
b. Place the strips on a paper towel on your lab bench. Using pencil, mark an orientation on
each strip: + and - at opposite ends and the application start position (▲) centered in the
middle of the strip. The circle (◌) represents where the sample will be spotted (DON’T
DRAW THE CIRCLE, it’s just where your sample will be spotted). Label each strip along one
edge either: SM (skeletal muscle), K (kidney), L (liver), H 4 , M4.
SM
+
◌
-
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c. In the cold room, pour ~150 mL pH 8.8 Barbital buffer into a clean tray large enough to hold
all 5 strips. Gently float each strip on the buffer, but do your best not to submerge. If there
are white spots on the strips, discard and get a new strip. Allow strips to soak for at least 10
min.
d. Mark an electrophoresis chamber with your group’s section and names.
e. Transfer cold barbital buffer to the electrophoresis chamber – fill only to the fill line on each
side. The buffer should not cross the center divider.
f. Without allowing buffer to spill onto the top of the strips, lay the strips across the holders,
orienting them with the “+” and “–“ ends of the chamber. Make sure they lay flat at the top
and do not sag. If buffer gets onto of the strip, gently dab with a paper towel to try to
remove as much buffer as possible without damaging the surface of the strip. . It is very
important that there is no excess buffer on the surface of the strips.
g. Place the lid on the chamber and connect to the power supply and set to 225 V constant
voltage. Press run and when the voltage reaches 225V, check the current by pressing
Current. It should be at 0.75 - 2 mA per strip (multiply by 5 if 5 strips, or 10 if 2 groups use
the same power supply). If the current does not fall within these parameters, consult with
your TA. Turn off the power supply and disconnect before proceeding to the next step.
5) Running the CAE – done in the cold room
a. Remove the lid from the chamber and load ~1.5 µL of each sample on the appropriate strip.
Remember that you do not want the strips to sag in the middle. Touch the strip lightly to
add the sample. Let soak. You get better results if you allow to “dry” (more like soak into
the strip) for a few minutes, but only do this if you have extra time.
b. Replace the lid, reconnect to the power supply and run at 225 V. Be sure to check that the
voltage reaches 225 V and the current is correct before leaving the cold room.
c. Run for 1.5 – 2 hr. Check periodically to make sure it is running. Prepare the staining mix at
the end of the run (see step 5).
d. Unplug your apparatus from the power supply. Sometimes this creates a change in current
which turns shuts down the circuit. Be courteous and make sure your classmates’
experiment is still running if you were sharing a power supply. If nobody else is using the
power supply, turn off the power supply and unplug.
5. Staining – complete at RT
a. Just before you turn off the electrophoresis, prepare the staining mix in a small white
chamber.
Mix in a 15 mL tube:
7 mL nitroblue tetrazolium (3 mg/mL)
0.7 mL phenazine methosulfate (3 mg/mL)
2.0 mL of NAD+ (50 mg/mL)
1.4 mL of 30% sodium lactate
(solution should be transparent; if cloudy, try mixing another)
b. Get 5 strips of Whatman paper and lay them on a paper towel on the bench top.
c. With forceps, dip the strips one at a time in the staining mix (sample side down) and then
place on Whatman paper (sample side up). The residual staining mix will be enough for the
reaction to proceed.
d. Watch for blue spots to develop. Then turn the strips over to soak up remaining dye to stop
reaction. There should be more than one spot for each crude extract. You may wish to wait
longer if you find fainter bands taking longer to develop.
6. Collecting Data
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a. Draw a diagram of each strip in your lab notebook and using a ruler, record the distance
the spots traveled from the center. Do this step quickly as the stain tends to fade over
time.
b. Place all 5 strips on a glass plate and photograph using the gel documentation system or
scanner. If using a scanner, be sure to cover scanner with saran wrap to avoid
contamination. You will want to save an electronic version of the photo on the desktop
(Chem108, Year and quarter, section#). Try to use a thumbdrive since internet
connection is often slow. Print out one copy for each student in the group to tape into
the lab manuals.
7. Clean up
a. Clean up light box and dispose of all strips and Whatman paper in the trash.
b. Return the glass plate to your TA
c. All Barbital buffer and staining solution waste should go in the waste container in the
cold room
d. Rinse out the electrophoresis chambers with DI water and leave to dry.
Questions to think about:
1. More than a single spot is found in several tissue samples. What advantage do isoenzymes
provide to an organism and to a single tissue with multiple isoenzymes?
2. What physiological differences are taken into account through different LDH isoenzyme
populations in kidney and muscle?
3. What electron transfer reactions occur with the staining procedure? What changes in the
structures of the involved molecules lead to the resulting blue color?
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Exercise 5 Purification of LDH by Precipitation with Ammonium Sulfate
Key Principles:
Purification of an enzyme from a specific organ; Centrifugation; Solubility of protein; Salting out;
Removal of small molecules by dialysis.
Description of Procedure:
Protein purification is a standard practice in biochemistry. One of the best ways to study a protein is to
isolate it from all other proteins and then conduct experiments. For example, you now know that if you
add NADH and pyruvate to your extract, the NADH appears to be reduced to NAD+. However, do you
know whether NADH and pyruvate are the only necessary components for the reaction? There are
many other proteins present in the tissue extract. Perhaps another protein in the extract also catalyzes
the same reaction and is contributing to the measured activity. If we purify away all other proteins and
still observe that the purified LDH catalyzes the conversion of NADH to NAD+ in the presence of
pyruvate, then we can be more confident that LDH is the protein that catalyzes the reaction.
Protein purification can be considered as an iterative process that contains two steps. The first step is
fractionation, or separation, of proteins. Fractionation is usually performed by taking advantage of the
differences in physical properties of the various proteins in the mixture. Some of these properties
include charge, size (molecular weight), hydrophobicity, chemical affinity, etc. Following fractionation,
you often end up with many samples. Some of the samples contain your protein of interest while others
contain contaminants. This leads us to the second step of protein purification which is analyzing the
fractionated solutions. The analysis used can be one of any number of methods such as enzymatic assay
and gel electrophoresis (we’ll learn more about gel electrophoresis later). Lastly, the protein of interest
is often not completely pure after a single purification step. The entire process can be repeated, i.e. it is
iterative, by applying the partially purified protein to another purification method and then analyzing
the fractionated samples.
In this experiment, bovine heart tissue will be homogenized and proteins will be selectively precipitated
out of solution using step-wise increases in the concentration of ammonium sulfate. Each step-wise
increase in salt concentration is often referred to as a “salt cut.” Both proteins and salt interact with
water molecules to remain soluble. At high concentration of (NH4)2SO4, water molecules that are
available to solvate the proteins becomes scarce. The result is a phenomenon known as “salting out,”
where proteins aggregate and precipitate out of solution. Different proteins salt out at different
concentrations of salt. After each salt cut, salted out proteins are separated from those remaining in
solution by centrifugation. In this fashion, proteins can be fractionated and purified away from each
other. Ammonium sulfate precipitation is a good first step for protein purification. It concentrates the
protein and the precipitated protein is very stable since (NH4)2SO4 is a stabilizing salt. The one
disadvantage is that it leaves the protein in a high salt concentration which may interfere with
subsequent analysis or purification procedures.
Following (NH4)2SO4 precipitation, the resulting fractions from each salt cut will be assayed for activity of
LDH. The fractions with the highest total activity (NOT activity concentration) of LDH are dialyzed to
remove the ammonium sulfate and other small molecules. Aliquots of fractions with the highest
enzymatic activity are saved for the next purification method.
LDH Exercise 5: Purification of LDH by Precipitation with Ammonium Sulfate
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Selected References:
Boyer, R (2006) Protein production, purification and characterization. In: Biochemistry Laboratory:
Modern Theory and Techniques, Pearson/Benjamin Cummings, pp 329-339.
Kyte, J. (1995) Structure in Protein Chemistry, Garland Publishing, New York, pp. 1-24. Scopes, R. K.
(1994) Protein Purification, 3rd ed, Springer-Verlag, New York, pp 17-21 & 76-85 Segal, I. H. (1976)
Biochemical Calculations, John Wiley and Sons, New York, pp. 287-290.
Materials:
In cold room: bovine heart tissue, weigh boats, triple beam balance, sharp knife or scalpel,
thermometer, chilled 50 mM sodium phosphate, 10 mM EDTA, pH 7.5 buffer (for homogenizing), Waring
blender, Teflon pestle, homogenizer and motor, dialysis tubing, clips for dialysis tubing, 20 mM sodium
phosphate, 10 mM EDTA buffer, pH 6.0 (for dialysis)
In lab: ice bucket with ice, 250 mL beaker, 100 mL graduated cylinder, glass stirring rod, two 250 mL
centrifuge tubes, Beckman J2 preparative centrifuge and JA-14 rotor, 1.5 mL eppendorf tubes, pipetman
and pipet tips, eppendorf tube rack, ammonium sulfate, analytical balance, weigh boats, stir plate, stir
bar 1 L beaker, glycerol, isopropanol/dry ice in dewar flask, -20°C freezer.
Safety: the solutions and tissues used in this lab are not toxic and can be disposed of down the drain.
Ammonium sulfate – avoid eye and skin contact; wash well with water in case of contact. Waste can go
in the trash, but attempt to minimize excess by sharing with other students.
Procedure: IMPORTANT! You will use information from this experiment to fill in Table Exercise 7-1.
Be sure to write down the pertinent information (especially volumes of your samples).
Day 1
1) In cold room, weigh approx. 40 g of chopped heart tissue – record mass accurately – using a
scale.
2) Add heart tissue to blender with 160 mL of 50 mM sodium phosphate, 10 mM EDTA, pH 7.5
buffer
a. Blend at 30s intervals
b. Check temp periodically and keep below 20°C
c. Transfer to 250 mL beaker and store on ice
3) In cold room, transfer 50 mL of blended tissue to homogenizer (save the remainder)
a. While holding the tube by the sides, homogenize until the pestle moves smoothly
through the extract and there are no chunks on the bottom.
b. Check temp periodically and chill on ice if >20°C.
c. When homogenized, transfer to new beaker and store on ice
4) Repeat step 3 with another 50 mL of blended tissue until entire sample is homogenized.
a. Combine homogenates.
5) Transfer entire homogenate to one 250 mL centrifuge tube.
6) Balance your tube with another group by adding chilled buffer
7) Centrifuge at 13,000 rpm for 20 min at 4°C in JA-14 rotor
8) While centrifuging
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a. Prepare 8 eppendorf tubes labeled as “heart crude extract,” the date, and your initials.
Place in a new eppendorf rack, labeled “Heart Extract – LDH Purification”
b. Clean up blender and homogenizer in cold room and have TA check your area
9) After centrifugation is complete, pour the supernatant into a 250 mL graduated cylinder in the
cold room and record the volume. This is “heart crude extract”. Discard pellet into the white
trash can in cold room, not the sink!
10) In one 15 mL Falcon tube (disposable plastic tube), add 5 mL of your heart crude extract and
gently mix with 500 L of glycerol. (Pre-chill the falcon tube to keep your sample cold. Do not
vortex). Aliquot 0.5 mL into each of the 10 prepared “heart crude extract” tubes.
a. Save the remainder of your heart crude extract for ammonium sulfate precipitation
11) Set aside one tube for an enzyme assay and store it on ice.
12) Quick freeze the remaining 9 eppendorf tubes in isopropanol/dry ice and store in a tube rack
labeled “Heart Extract” in -20°C Freezer
First Ammonium Sulfate Precipitation (40% cut) - done in cold room
13) Measure and record the volume of the remaining initial supernatant.
14) Pour the supernatant into a 250 or 400 mL beaker and add a stir bar. Place on a stir plate in the
cold room.
15) Calculate and weigh the amount of ammonium sulfate you will need. For every mL of
supernatant, you will require 0.226 g of solid ammonium sulfate.
16) Add ammonium sulfate slowly to the supernatant in the beaker on the stir plate over a 15 min
period with constant stirring. If added too quickly, the local concentration of ammonium sulfate
will become greater than 40% near the undissolved salt crystals causing some of the more
soluble proteins to inappropriately precipitate out and reduce the purity of this salt cut.
17) Allow to stir an additional 10 min to allow the ammonium sulfate to completely dissolve.
18) Either transfer to a 250 mL centrifuge bottle and balance with another group if your volumes are
close or transfer to two 250 mL centrifuge tubes and balance by moving solution from one
bottle to the other – do not balance by adding buffer.
19) Centrifuge at 13,000 rpm for 20 min at 4°C.
20) While centrifuging, prepare 7 eppendorf tubes labeled as, “40% supernatant,” the date, and
your initials and 7 eppendorf tubes labeled as “40% pellet,” the date, and your initials.
21) In the cold room, pour the supernatant into a clean graduated cylinder and record the volume.
Save the pellets on ice.
22) In one 15 mL Falcon tube (disposable plastic tube), add 3.5 mL of your “40% supernatant” and
gently mix with 350 L of glycerol. (Pre-chill the falcon tube to keep your sample cold. Do not
vortex.) Aliquot 0.5 mL into each of the 7 prepared “40% supernatant” tubes.
23) Set aside one tube for an enzyme assay and store it on ice.
24) Quick freeze and store in “Heart Extract” Rack in -20°C Freezer.
25) Store the 40% ammonium sulfate pellets and supernatants in labeled tubes in the cabinet
assigned to your section in the cold room. Do not freeze! Be sure to clean up. Do not leave ice
buckets/stir plates in cold room.
26) Redissolve your pellet(s) in a total of 20 mL of cold 50 mM sodium phosphate, 10 mM EDTA, pH
7.5 buffer. Tap gently against your hand or pipet up and down (avoid frothing).
27) Repeat step 22 for the “40 % pellet” tubes, aliquot 0.5 mL of resuspended 40% pellet into seven
tubes, quick freeze six tubes and store in -20°C Freezer. Set aside one tube for enzyme assays and
store it on ice.
28) Measure the volume of the resuspended 40% pellet and record. Save the resuspended pellet on
ice or in the cold room.
Day 2
LDH Exercise 5: Purification of LDH by Precipitation with Ammonium Sulfate
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Second Ammonium Sulfate Precipitation (70% cut) – This may be completed on the same day as the first
ammonium sulfate precipitation if there is time. See your TA before beginning. If you stop here, be sure
to save your 40% supernatant, 40% pellet and samples reserved for enzymatic assays at 4oC, do not
freeze.
29) Measure and record the volume of remaining 40% supernatant.
30) Pour the supernatant into a 250 – 400 mL beaker and add a stir bar. Place on a stir plate in the
cold room.
31) Calculate and weigh the amount of ammonium sulfate you will need. For every mL of
supernatant, you will require 0.187 g of solid ammonium sulfate.
32) Add ammonium sulfate slowly to the supernatant in the beaker on the stir plate over a 15 min
period with constant stirring.
33) Let stir an additional 10 min – ammonium sulfate should be completely dissolved.
34) Transfer to two 250 mL centrifuge bottles and balance by moving solution from one bottle to
the other – do not balance by adding buffer. You can attempt to balance with another group if
your volumes are close.
35) Centrifuge at 13,000 rpm for 20 min at 4°C in JA-14 rotor.
36) While centrifuging, prepare 7 eppendorf tubes labeled as, “70% supernatant”, the date, and
your initials; and 4 eppendorf tubes labeled as “70% pellet”, the date, and your initials.
37) In the cold room, pour the supernatant from both bottles into a clean container. Save the pellets
on ice.
38) In one 15 mL Falcon tube (disposable plastic tube), add 3.5 mL of your “70% supernatant” and
gently mix with 350 L of glycerol. (Pre-chill the falcon tube to keep your sample cold. Do not
vortex.) Aliquot 0.5 mL into each of the 7 prepared “70% supernatant” tubes.
39) Set aside one tube for an enzyme assay and store on ice.
40) Quick freeze 6 tubes and store in “Heart Extract” Rack in -20°C Freezer.
41) Measure the volume of the remaining supernatant using a graduated cylinder.
42) If you stop here, store the 70% ammonium sulfate pellets and supernatants in labeled tubes in
the cabinet assigned to your section in the cold room. Do not freeze! Be sure to clean up. Do
not leave ice buckets/stir plates in cold room.
43) Re-dissolve pellets in a total of 15 mL of cold 50 mM sodium phosphate, 10 mM EDTA, pH 7.5
buffer. Tap gently against hand or pipet up and down avoiding bubbles.
44) Measure volume of re-dissolved pellets and record.
45) Pipet 1.5 mL of dissolved 70% pellet in one Eppendorf tube. Add 150 L of glycerol, mix by
pipetting and then aliquot 0.5 mL into three Eppendorf tubes labeled “70% pellet.”
46) Quick freeze and store in “Heart Extract” Rack in -20°C Freezer.
47) Aliquot 0.5 mL of 70% pellet into 2 tubes for enzyme assays and store at 4°C.
Assaying the LDH Fractions
48) Assay as done in LDH Ex. 2 – first optimize each sample by finding the correct dilution to yield a
decrease of 0.1-0.2 A340 units per minute, then repeat for 2 good trials. You cannot assay any
ammonium sulfate fractions that have been frozen as they will have lost activity – use the
material stored in the cold room.
49) Samples to assay: heart crude extract, 40% supernatant, 40% pellet, 70% supernatant, and 70%
pellet
50) Multiply the assay activity (µmol/min/mL) by the total volume of the fraction to determine the
total activity of the fraction (µmol/min).
51) The majority of your activity should be in the 70% pellet. If it is in the 70% supernatant you will
need to complete an 85% ammonium sulfate precipitation adding 0.413 g of ammonium
sulfate/mL of extract and proceeding as done for the 40% and 70% cut.
LDH Exercise 5: Purification of LDH by Precipitation with Ammonium Sulfate
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Dialyzing – your TA will demonstrate setting up the dialysis
52) Obtain a 1 L or 0.8 L beaker and fill with 20 mM sodium phosphate, 10 mM EDTA, pH 6.0. Make
sure that you use the correct buffer!
53) Place a stir bar in the bottom and place on a stir plate in the cold room.
54) Prepare dialysis tubing by twisting one end and tying a knot.
55) Test the integrity of the knotted dialysis tubing by adding a small volume of cold dialysis buffer.
Check for leaks, continue if there is no leakage and remove the buffer.
56) Use a Pasteur pipet to transfer the fraction with the most total activity into the dialysis tubing.
Consult with your classmates. Everyone should draw the same conclusion about which fraction
had the most total activity.
57) Leave a small amount of room for the volume to increase (water in the buffer will enter the
tubing to dilute the ammonium sulfate), twist and clamp the other end of the tubing with a
clip, if necessary add a second clip adjacent to and in the opposite orientation of the first
clip.
58) Place the dialysis tubing with the sample in the buffer and place in the cold room.
59) You will need to come in to change the buffer one times over the next 24/48 hrs. With each
change of the buffer, the ammonium sulfate concentration will become further reduced. Your
TA will arrange a time for you to exchange the dialysis buffer. To change the buffer, pour old
buffer in the sink in the cold room and refill the beaker with fresh buffer.
60) Depending on your section, your dialysis buffer may be moved from the cold room countertop
to the cabinet below if another section needs the space.
Clean up
61) Pour all ammonium sulfate solutions down the drain except for those that you are dialyzing. Do
not store them in the cold room.
62) Wash all glassware and clean up the cold room. There should be no meat products in the sinks
or trash in the labroom.
Intended Use of the Heart Crude Extract Aliquots
Four: Enzymatic Assay/Bradford (Exer 7)
Two: Native PAGE (Exer 8)
One: SDS-PAGE (Exer 9)
One: Western blot (Exer 13)
Questions to think about:
1. LDH is functional in mammals at 37oC. What are the reasons for keeping samples at or near 4oC
during our manipulations?
2. Why is a low concentration of salt necessary to keep many proteins in solution, while a higher
concentration of salt causes precipitation of these same proteins?
3. What is occurring during dialysis? Can you think of another technique that might be used to
secure similar results?
4. What is the purpose of using consecutive ammonium sulfate precipitations?
LDH Exercise 5: Purification of LDH by Precipitation with Ammonium Sulfate
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Exercise 6 Purification of LDH by Affinity Adsorption to BioSepra Blue
Trisacryl MR
Principles: Purification of LDH by column chromatography, affinity chromatography
Background:
Protein purification is an iterative procedure. Following one step of purification, subsequent purification
methods take advantage of orthogonal properties of the protein. In Exercise 5, you took advantage of
the different solubilites of proteins in the presence of ammonium sulfate to separate LDH from
contaminating proteins. In this experiment, you will take advantage of LDH’s inherent affinity for a
chemical known as Cibacron Blue.
Cibrcron Blue is a blue colored dye that happens to have a relatively high affinity to proteins that use
NAD+/NADH as cofactors. This molecule can be covalently linked to inert beads, often referred to as
resins. Once linked to beads, the resin is placed inside of a column which is typically a glass tube with a
porous plastic bottom piece, known as a frit. The frit keeps the affinity resin inside of the column, yet
allows buffer to pass through and escape from the column. Many resins have a certain binding capacity,
meaning that there is an upper limit of protein that can be bound to the affinity molecule. The binding
capacity gives a guide for determining how much resin to add to the column. Once this volume is added
this amount of resin is referred to as the bed volume or column volume.
Purification using nearly all resins consists of the same steps. The resin is equilibrated by adding buffer,
usually the buffer that the protein is in. Next the protein solution is applied to the resin to allow the
protein of interest to become immobilized after binding to the affinity resin. Other proteins will flow
through the column. Then the resin is washed in buffer, which is usually the same buffer as that used
for equilibrating. Finally the protein of interest is eluted using a buffer that disrupts the interaction
between the protein of interest and the affinity tag. The disruption may occur as the result of changing
the pH or by adding a reagent that competes with the interaction between the protein and the affinity
resin. Since it was stated that Cibacron Blue binds to proteins that use NAD/NADH + cofactors, it would
make sense to use NAD or NADH+ to elute LDH. This procedure should prove effective, however it
would also be expensive since we would have to use large volumes of NAD or NADH+. Instead, we will be
disrupting the interaction using high salt concentrations. Since LDH can be eluted with high salt
concentrations, the interaction between LDH and the resin is ionic in nature. It turns out that this resin
binds other proteins in addition to LDH so we will be eluting with increasing salt concentrations to
further fractionate the proteins.
As part of this purification step, you will need to determine which elution fraction contains LDH. If you
discover LDH is primarily located in the flow through, then LDH did not bind to the column. If it is found
in the wash, then some component in the wash buffer caused the protein to elute. If everything goes as
planned, then LDH will be located in one of the high salt elution fractions. Thus far, you have been
monitoring LDH via enzymatic assay. Often, this type of experiment results in a large number of tubes to
assay, making the enzymatic assay unfeasible to complete in a reasonable amount of time. Instead, you
will initially use a different assay, known as A280. Conjugated pi electrons found in aromatic molecules
can absorb light at 280 nm. Since proteins contain amino acids that have aromatic side chains
(phenylalanine, tyrosine, and tryptophan), solutions that contain protein will absorb light at 280 nm.
Thus an A280 assay gives a measure of total protein content. It is most useful because it is very quick; all
LDH Exercise 6: Purification of LDH by Affinity Adsorption to BioSepra Blue Trisacryl MR
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you need to do is place the sample in a UV compatible cuvette and take an absorbance reading at 280
nm. You should keep in mind that this assay cannot distinguish between LDH protein and contaminating
proteins so it is not a substitute for the LDH enzymatic assay. Instead, you will use the A 280 assay to
monitor which fractions contain protein to help narrow down which tubes you should assay for
enzymatic activity.
Clamp
Blue Trisacryl M packed in column
Waste beaker
Figure 11 Experimental setup for column chromatography.
Selected References:
Boyer, R (2006) Affinity chromatography and immunoadsorption. In: Biochemistry
Laboratory: Modern Theory and Techniques, Pearson/Benjamin Cummings, pp 163-173. Cuatrecasas, P.
(1970) Protein Purification by Affinity Chromatography. J Biol Chem
245:3059-3065.
Everse, J., and Kaplan, N.O. (1973) Lactate Dehydrogenase: Structure and Function. Advances in
Enzymology, vol 37 (Meister, A., ed.), John Wiley and Sons, New York, pp. 61-133. Kaplan, N.O. et al.
(1974) Purification and Separation of Pyridine Nucleotide-Linked
Dehydrogenases by Affinity Chromatography Techniques. Proc. Natl. Acad. Sci. 71:3450-3454
Kyte, J. (1995) Structure in Protein Chemistry, Garland Publishing, New York, pp. 1-24.
Scopes, R. K. (1994) Protein Purification, 3rd ed, Springer-Verlag, New York, pp 8-14, 187-204. Segal, I. H.
(1976) Biochemical Calculations, 2nd ed, Wiley Press, New York, pp. 324-346.
Indiana University Chem (2011) Affinity Chromatography for the Isolation of LDH.
http://courses.chem.indiana.edu/s117/documents/Ch5AffinityChromatography.pdf
Materials:
In cold room: Dialyzed AS LDH heart fraction; Blue Trisacryl M (slurry in buffer); 2 M NaCl in 0.02M
sodium phosphate buffer (pH 6.0), 10 mM EDTA; 100 mM sodium pyruvate; dialysis tubing;
In -20°C freezer: 15 mM NADH
In lab: 38 mL screw cap centrifuge tubes; JA-14 rotor and centrifuge; cheesecloth; Funnel; 50 mL
graduated cylinder; 10 cm plastic column for chromatography; ring stand; clean beakers (assorted);
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Pasteur and graduated pipets; P20, P200, P1000 and tips (for assays); 100 mM phosphate buffer, pH 7.5;
500 mM phosphate buffer, pH 7.5; Spec 20; Eppendorf tubes; glass culture tubes; eppendorf tube and
culture tube racks; UV-Vis Spectrophotometer;1.5 mL cuvettes; stirring plates
Safety: The solutions used are nonhazardous and can be disposed down the drain.
Time management guideline
If you find yourself running behind schedule, you can use a syringe to push the sample through the
column. This tends to compact the resin, though and the column will tend to flow a lot slower without
your assistance (meaning you will probably have to sit next to the column and continually press the
syringe for the rest of the column run). Be careful when using the syringe. Too much pressure can crush
the resin. A reasonable flow rate is ~1 mL/min.
(60 min) Load sample and collect the flow through
(90 min) Wash the column
(60 min) Collect elution fractions
(60 min) Complete assays, prepare samples for storage
Many of the assays and sample preparation procedures can be done while washing the column
or eluting the protein
Procedure:
Important: Biochemists will usually perform column chromatography of enzymes in the cold room or in
a deli fridge. We don’t have enough space for everyone in the cold room. As a compromise the column
is run at RT, but the buffer and the samples before and after chromatography should be kept on ice.
Also, you will use data for this experiment for Table Exercise 7-1 in exercise 7. Be sure to write down
information for this table.
1) One student from each group should work on preparing the sample for chromatography (step 2)
while the other can work on setting up the column (step 3).
2) Sample Preparation
a. Remove the dialyzed heart extract from the dialysis tubing using a pipet and transfer to
a 38 mL screw cap centrifuge tube on ice.
b. Balance tubes and centrifuge at 13,000 rpm for 15 min at 4°C in JA-14 rotor.
c. Pour supernatant through cheesecloth set in a funnel to remove particulate matter.
d. Measure and record the volume using a graduated cylinder.
e. Aliquot five 0.3 mL fractions into Eppendorf tubes labeled “precolumn dialysate” Keep
one fraction on ice and assay as in Ex. 2 – start with 50 µL, dilute as necessary and
repeat for 3 good assays. You may wait and perform the assay on the dialysate/column
load when you assay the flow through. Trim the end off of a P200 pipette tip and add
25 l glycerol, and mix, to the remaining aliquots and freeze using dry ice/isopropanol.
3) Prepare for Chromatography (This may be done for you in advance)
a. Connect a 10 cm column to a ring stand using a two pronged clamp. Make sure that the
column is vertical and the stopcock is closed. Place a 250 – 400 mL beaker under
column to collect waste. Raise the column until a test tube rack with 13 x 100 mm
culture tubes can be set below the column.
b. Fill the column halfway with 20 mM phosphate buffer, pH 6.0, 10 mM EDTA.
c. Resin in the stock bottle will have settled to the bottom. Swirl Blue Trisacryl M
thoroughly to resuspend the resin and transfer about 6 mL of this slurry into your
column using a 10 mL graduated pipet. Open the stopcock and allow the Blue Trisacryl
M to settle while the column is flowing (buffer should collect in the waste beaker
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below). Add more buffer as needed to keep bed from drying out while being careful not
to disturb bed. Blue Trisacryl M should settle into a fairly flat column about 3 inches in
height.
d. Run about 10 mL of buffer through the column to pack the column. Collect the buffer in
the waste beaker.
4) Loading the ammonium sulfate dialysate onto the column.
a. Place ten 100 x 13 mm culture tubes in a test tube rack and number them FT-1, FT-2,
etc. to FT-10 (FT for flow through). Pipet 5 mL water into an unlabeled test tube for use
as a reference to identify the 5 mL level.
b. Let the buffer in the column drain into the waste beaker until the level is just above
surface of the column. Stop the flow using the stopcock.
c. Carefully fill the column completely with half of the remaining heart extract by pipette
while trying not to disturb the bed of resin. Once the column is filled, place the first test
tube under the column to begin collecting the flow through and open the stopcock.
Note: you will only load half of your LDH extract to save time and to avoid exceeding the
binding capacity of the column. The first 3 mL of the flow through will be equilibration
buffer.
d. After 5 mL has drained into the first test tube, move the rack over so that the column
elutes into the second test tube. As the column drains, add more heart extract to the
top, being careful not to disturb the bed of resin. If the column clogs, you may need to
apply some of pressure – consult with your TA. A faster flow rate will result in poorer
binding to the column.
e. While the column is running, assay the flow through fractions for LDH activity as done in
Exercise 2. This will tell you whether LDH is binding to the column efficiently. Do not be
surprised if there is some activity – but it should not be as high as the dialysate/column
load. Record the activity in your notebook. If it is high (>10% of initial) – check with
your TA.
f. When the entire extract has been loaded on the column, allow the liquid level to drain
to just above the bed of the column. Add approximately one bed volume of 20 mM
phosphate buffer (pH 6.0), 20 mM EDTA and allow the liquid to drain again. Turn off the
flow with the stopcock at bottom of column. Do not allow column to run dry. Store the
flow through sample on ice.
g. Obtain two eppendof tubes and place 100 L of the second to last flow through fraction
into each. Label and store in the -20oC freezer for your gel electrophoresis experiment.
You do not need glycerol and you do not need to flash freeze.
h. Pool the rest of your flow through, measure the volume, and take an accurate activity
measurement. Discard if there is little activity. Check with your TA if the activity is high
compared with your starting material.
5) Wash the Column
a. Use a graduated cylinder to obtain 30 mL of 20 mM phosphate buffer, pH 6.0 and set it
in a beaker on ice. Prepare tubes for collecting the wash. Label them W1 through W7
(W for Wash) and place them in a test tube rack under the column.
b. Carefully, without disturbing the bed of resin, fill the column chamber with the buffer
and open the stopcock, starting the wash. (The first 2-3 mL that flows out the column
will be the remaining ‘flow through’).
c. Wash with 30 mL, collecting 5 mL fractions. Fill the column with buffer as it drains.
Keep liquid in the column near the top to increase the flow rate.
d. While the column is running, you can begin the A280 assay (see step 7).
e. When 30 mL have passed through the column and A280 is near zero, close the stopcock
at bottom of the column. If the A280 is still high, check with your TA.
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6) Elution of LDH
a. Keep in mind that purified LDH should come off the column in these fractions. Move
tubes to ice or cold room when filled. Proteins will elute from the column at higher
ionic strength (salt concentrations). You will elute protein from the column using 10 mL
each of 0.05, 0.25, 0.50 and 1.00 M NaCl Sodium Phosphate, pH 6.0
b. Label four 15 mL falcon tubes with the 4 different salt concentrations.
c. Carefully fill column with 10 mL of 0.05 M NaCl/sodium phosphate and open stopcock to
begin collecting fractions – 10 mL each.
d. Repeat step b with the rest of the buffers.
e. Measure the A280 (described in step 7)
7) A280 Assay. Most plastics and glass will absorb light at 280 nm and are not acceptable materials
for the A280 assay. The cuvettes we will be using are made of a special material. They are
disposable but somewhat expensive. Please rinse and reuse them for the wash samples and
discard when finished for all wash samples. Obtain a fresh cuvette for elution samples. While
the column is washing, one student from the group can begin measuring the A 280 of each
fraction beginning with the first wash until the final elution. If the spectrophotometer station
gets too crowded, the measurements can also be done at the end of the each step (washing,
eluting).
a. Each of the specs in the room is slightly different so check with your TA before using.
b. Set the UV-Vis Spec to 280 nm.
c. Blank the UV-Vis Spec with 1 mL of buffer (should be 20 mM phosphate, pH 6) in the
cuvette.
d. Be careful not to touch the window of the cuvette where the light will pass through.
e. Fill the cuvette with 1 mL of wash or elution fractions.
f. Place the cuvette in the spectrophotometer, paying attention to the cuvette orientation
such that the light in the spectrophotometer passes through the cuvette’s window.
g. Measure the A280 and record. Return the solution to the test tube. Do not discard after
measuring. Save all fractions on ice or in the cold room until the end of the lab period.
h. For the wash fractions, the A280 should decrease to near zero by the end of 30 mL. If it
does not, check with your TA.
i. For the elution fractions, the A280 should be higher for certain fractions and lower for
others.
8) LDH enzymatic activity assay
a. Measure the LDH activity of each elution fraction, the flow through, and the precolumn
dialysate as performed in Exercise 2.
b. Determine which of the elution fractions has the highest LDH activity. Note: you must
pay attention to the dilution and the volume of elution that you add to the assay. For
example, if 50 L of a 1/10 dilution from the 0.05 M salt elution is added to the assay
and has an dA/dT of 0.2, while 5 L of a 1/1000 dilution from the 0.25 M salt elution is
added to the assay and has a dA/dt of 0.1, the 0.25 M salt elution has more LDH even
though the dA/dt is less.
c. One (or maybe two) of the elution fractions should a have much greater LDH activity
than the others.
d. Aliquot 6 mL of each elution fraction into pre-chilled falcon tubes. Mix in 600 L of
glycerol by pipetting or inverting. Aliquot 1 mL of each sample into 6 Eppendorf tubes.
Their labels should include their corresponding salt elution concentrations (i.e. 0.05 M,
0.25 M, 0.50 M, 1.00 M) Add glycerol to ~10% (v/v) and quick freeze in a dry
ice/ethanol bath.
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9) Clean Up. Do not discard any solutions until you have identified the location of your LDH
enzymatic activity and have set up the dialysis. All washes can go down the drain. Close the
stopcock on the column and fill the column with phosphate buffer before leaving for the day.
Intended Use of the Postcolumn Pool Dialysate Aliquots
One: enzymatic assay (if not done immediately after dialysis step)
One: Protein assay
One: Native Gel (Exer 8)
One: SDS-PAGE (Exer 9)
One: Western (Exer 13)
One: backup
Questions to think about
1. Why was ammonium sulfate precipitation used before the affinity column?
2. Blue dextran is not a substrate of LDH. What properties allow it to bind LDH
specifically? What other molecules could be added to a column matrix to elicit similar binding?
3. What allows the salt to dislodge LDH from the column?
4. What are some other possible methods to elute LDH from the column?
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Exercise 7 Protein quantification assays: Bradford and UV-absorption assays
Key Principles:
Bradford assay for determination of protein; Use of A280 and protein extinction coefficient; Table of
protein purification; Activity concentration and specific activity; Turnover number; Yield of activity;
Degree of purification.
Background:
The goal of protein purification is to separate the protein of interest away from all other proteins. Thus
far, we have fractionated the proteins using various protein purification methods (i.e. ammonium
sulfate precipitation and Cibacron Blue affinity chromatography). A couple of obvious questions should
come to mind regarding these methods. One question is, “How effective is each purification method?”
and, “Which method is better?” To address these questions, we need to determine a method to
quantify the purification at each step.
Let’s consider how you might go about quantifying purification of LDH. For an impure LDH solution, the
ratio of LDH to total protein content will be low. For a pure LDH solution, the ratio of LDH to total
protein content will reach a maximum value. Therefore, as a solution becomes increasingly pure, the
ratio of LDH to total protein content will increase. Measuring and comparing this ratio following each
purification procedure provides a quantitative method for comparing the purity of LDH at each step.
The ratio that biochemists use to compare the purity of an enzyme sample is known as specific activity.
Specific activity is defined as follows.
𝑈𝑛𝑖𝑡𝑠
µ𝑚𝑜𝑙𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑓𝑜𝑟𝑚𝑒𝑑
𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝑚𝑔 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 𝑚𝑖𝑛 ∗ 𝑚𝑔 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛
Eq. 7-1
You already know how to obtain the numerator value for specific activity. It is simply the activity
measured by the LDH enzymatic activity assay. The denominator term is also something that may seem
somewhat familiar. In Exercise 6, you used an A280 assay as a measurement of total protein content.
The A280 is useful as a protein quantitation assay since it is extremely fast. We will not be using it in this
exercise due to its limitations, however. As you should recall, protein absorbance at 280 nm results
from the presence of aromatic groups on phe, tyr, and trp. The frequency of these amino acids varies
widely from protein to protein, making A280 a somewhat poor method for accurately measuring protein
content of a mixture of proteins. A280 measurements of protein concentrations for impure protein
mixtures are sometimes used to roughly estimate the total protein content of a solution by assuming
the average extinction coefficient for proteins is 1 Absorbance Unit = 1 mg/mL. A 280 is a much better
assay for quantifying a solution of pure enzyme after calculating the theoretical extinction coefficient
based upon the protein’s amino acid sequence.
In this experiment you will be using the Bradford assay, which is a common assay used for quantifying
protein content. This assay relies on binding of a dye, Coomassie Brilliant Blue G-250, to proteins. This
dye exists in two forms with different absorbance spectra. The binding of the dye to protein causes a
shift in its absorption maximum from 465 nm to 595 nm. Therefore, when Bradford reagent is used,
protein concentration is correlated with the absorbance detected at 595 nm. The protein concentration
LDH Exercise 7: Protein quantification assays: Bradford and UV-absorption
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of the unknown is estimated using a standard curve using an inexpensive representative protein like
bovine serum albumin (BSA) or bovine immunoglobulin (IgG).
Selected references:
Boyer, R (2006) Measurement of protein solutions. In: Biochemistry Laboratory: Modern Theory and
Techniques, Pearson/Benjamin Cummings, pp 69-74.
Bradford Assay, Specific Activity and Purification Summary Table Bio-Rad (2003) Protein quantitation:
Quick start Bradford protein assay. Tech bulletin 2969. http://www.bio-rad.com
Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-54
Kyte, J. (1995) Structure in Protein Chemistry, Garland Publishing, New York, pp. 1-24. Layne and Eunis,
“Spectrophotometric and Turbidimetric Methods for Measuring Proteins.”
Reprint from: Proteins and Derivatives. pp. 447-454. Miller, G.L. (1959) Anal. Chem. 31:964.
Segal, I.H. (1976) Biochemical Calculations, 2nd ed, Wiley Press, New York, pp. 287-290. Scopes, R. K.
(1994) Protein Purification, 3rd ed, Springer-Verlag, New York, pp. 44-50.
A280 Assay
Gasteiger E et al. (2005) Protein Identification and Analysis Tools on the ExPASy Server. In: The
Proteomics Protocols Handbook, John M. Walker (ed), Humana Press. pp. 571-607
Hoffman NE and Hanson AD. (1986) Purification and properties of hypoxically induced lactate
dehydrogenase from barley roots. Plant Phisiol 82:664-670.
Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. (1995) How to measure and predict the molar absorption
coefficient of a protein. Protein Sci 4(11): 2411-23.
Voet, D, Voet, JG and Pratt, CW (2008) Protein concentrations can be determined by spectroscopy.
Materials:
In cold room: 1 mg/mL IgG
In -20°C freezer: samples for assays (thaw completely before using). Do not stand with freezer door
open – take out your rack, close the door and select your samples. The rack will keep the samples cold
while you are sorting them.
In lab: Bradford staining reagent; P20, P200, P1000 and tips (for assays); 100 mM phosphate buffer, pH
7.5 (for dilutions); vortex; Spec 20; Eppendorf tubes; glass culture tubes; culture tube racks; UV-Vis
Spectrophotometer; cuvettes; stirring plates
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Safety: Bradford reagent contains phosphoric acid and must be handled with care and disposed of in
proper waste container. It is a general protein stain and, therefore, will stain skin and clothing.
Procedure:
Bradford assay
1) Turn on the Spec20 and set it to 595 nm. Let it warm up > 15 min.
2) Prepare the Standard curve
a. Standard concentrations will be 0, 15, 30, 45, 60, 75, 90 µg of IgG in 3 mL. (i.e. 0g/3mL,
15g/3mL, etc.), but 2.70 mL will be Bradford reagent. Therefore, you will need to mix 7
tubes at the appropriate concentrations: 0 mg/mL IgG, 0.05 mg/mL (15 g/300 L) IgG,
0.1 mg/mL (30 g/300 L) IgG, etc.) so that the correct mass of IgG is added to the
assay.
i. For example: To make the 15 µg sample, add 15 µL of 1 mg/mL IgG and 285 µL
of phosphate buffer to Bradford Reagent.
3) Prepare the heart extract samples
a. Samples to assay: heart crude extract, 40% supernatant, 40% pellet, 70% supernatant,
70% pellet; precolumn dialysate, 85% supernatant and pellet (if done), and saved
Cibacron Blue elution fractions and flow through.
b. To determine the concentrations of these samples, they will need to fall in the range of
the standard curve, that is, you need to add between 15 and 90 g of protein. Many of
your samples will need to be diluted, however, there is no way of knowing exactly how
much to dilute. Luckily, you can make a ballpark estimate based upon the A 280 reading.
Remember that an absorbance of 1.0 ~ 1 mg/mL of protein.
c. For example, if A280 = 0.305, then the concentration is ~0.305 mg/mL or 0.305 µg/µL.
Therefore to have 30 µg sample use 98 µL and dilute using 202 L of buffer. You may
wish to include other dilutions for each sample to help ensure that at least one dilution
falls on the standard curve
4) Assay all samples and standards
a. The color change of the Bradford dye once bound to protein takes time to develop and
changes over time. You should mix the Bradford assays for the standards and samples
all at once. It is considered bad practice to mix the Bradford with the standards, make
the sample solutions, then mix the Bradford with the samples. This also means that if
you don’t get the dilution correct for one of your samples, you should remix new
samples and a new set of standards.
b. Count the number of standards and sample conditions
c. Label that number of glass tubes and add 2.7 mL of Bradford reagent to each tube
d. Add 0.300 mL of the appropriate standard or sample to the tubes and immediately mix
by vortex.
e. Incubate for 5 min at RT. (You can determine whether your samples have concentrations
within the range of the standards by comparing the colors of the mixed samples/Bradford
reagent to the colors of the standards/Bradford reagent)
f. Blank the Spec20 with buffer (2.7 mL Bradford reagent and 0.3 mL of phosphate buffer).
g. Read and record the A595 for the standards and samples.
h. If any samples are not within the range of the standards, they will need to be repeated
with another dilution along with another set of standards.
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i.
Consult with your TA if you are running short on time to get permission to skip repeating
the standards and promise that you won’t take this type of short-cut if you find yourself
doing a Bradford as part of your job.
5) Clean up
a. Dispose of all Bradford reagent waste in the Bradford waste container.
b. All glass culture tubes can go into the glass waste containers.
c. Discard all samples except A280 postcolumn dialysate. This should be saved and frozen.
Handling the Data:
1) Use the Bradford Data to Calculate Protein Concentrations.
a. Plot a standard curve of A595 on the y-axis and g of protein on the x-axis. Draw a
smooth curve through the data. This is best performed in Excel, where you should
calculate the linear regression, determine the representative equation, and the Rsquared value.
b. Interpolate to determine the g of protein in each diluted sample from your
purification. Use the linear regression equation from the Excel plot.
c. Calculate the concentration of protein in each of your samples (in mg mL-1). Remember
to take into account dilution factors from mixing the assay and from diluting the
samples.
d. Calculate the total protein content for each sample
e. From your previous experiment, input the activity concentration and calculate the total
activity for each sample
f. Calculate the specific activity from the activity and protein concentration columns or the
total activity and total protein columns
g. Calculate the fold purification and % recovery for each purification step.
Table Exercise 7-1 Purification Table. For a worked example see Hoffman and Hanson (1986).
prot conc
activity conc
(undiluted)
(mol min-1 mL-1) (mg/mL)
Vol
(mL)
total activ
Total spec active
(mol min-1) (Prot (mol min-1 mg-1)
mg)
fold
purif
%
recovery
Heart crude
extract
40% sup
40% pellet
70% sup
70% pellet
precolumn
0.05 M elution
Flow through
0.25 M elution
0.50 M elution
1.00 M elution
2) Calculate the turnover number of your purified LDH. The turnover number is the moles of
product formed per second per mole of active sites. Its units are s-1. Assume that there are
LDH Exercise 7: Protein quantification assays: Bradford and UV-absorption
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36,724 g protein per mole of active sites in bovine heart LDH. This is the predicted molecular
weight of one bovine heart LDH monomer.
3) You will want to compare your result for the turnover number to those reported in the
literature. When looking for this value in Biochemistry by Berg, Tymoczko and Stryer, the
chapter by Holbrook (1975), or the article by Everse and Kaplan (1973), be aware that in some of
the older articles turnover number is referred to as Vmax and the unit may be min-1 rather than
s-1. Also consider variation in the reported values due to isoform differences and the species
from which the protein was purified.
Question: Why is it important to calculate the specific activity in addition to the total activity for each
step in the purification?
Acknowledgement: The procedure for the Bradford assay was kindly provided by Garth Ringheim.
LDH Exercise 7: Protein quantification assays: Bradford and UV-absorption
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Exercise 8 Electrophoresis of Native Proteins on Polyacrylamide Gels
Key Principles:
Demonstration of protein purity; Sieving effect of polyacrylamide on electrophoretic mobility; Effect of
the polyacrylamide concentration on sieving; Separation of native proteins on the basis of size, shape,
and charge; Discontinuous buffer system; Ionic boundaries and stacking effect; Coomassie stain vs.
Activity stain
Roles of acrylamide, bisacrylamide, N, N, Nˊ,Nˊ-tetramethylethylenediamine (TEMED) and persulfate
anion in gel polymerization.
Description of Procedure:
LDH will be analyzed by electrophoresis using a polyacrylamide gel as the matrix. Polyacrylamide is a
polymer made from repeating units of acrylamide. The reaction is initiated by a free radical formation
generated after the adding ammonium persulfate (APS) and tetramethylethylenediamine (TEMED).
Individual chains of acrylamide are linked together using a small amount of bisacrylamide. This creates a
chemical 3-dimensional webbing or mesh where the average pore size is controlled by the amounts of
acrylamide/bisacrylamide that are added to the polymerization reaction.
Following polymerization, the mesh becomes a pliable solid or gel. The pore size of the mesh is small
enough to impede the movement of most proteins. When an electric current is applied to the proteins
in the gel, the proteins are separated from one another into distinct bands due to different migration
rates. The charge to mass ratio plays a role as described in Exercise 4. When polyacrylamide is used as a
matrix, though, it allows further separation due to a sieving effect. The meshwork physically impedes
larger proteins while smaller proteins are able to pass more freely. Since mass is related to how large or
small a protein is, the sieving effect enhances the separation due to these physical properties. An
additional result of the sieving effect is that molecules can be separated by their shapes as well. You
may have come across this characteristic if you’ve taken the recombinant DNA course and saw that
supercoiled DNA migrates faster than linear DNA or relaxed circular DNA in agarose gel electrophoresis.
If this does not sound familiar to you, you need not consider it further except that know that shape
affects protein migration in polyacrylamide gels. Elongated proteins will tend to migrate slower than
globular proteins.
Polyacrylamide gel electrophoresis (PAGE) is a standard method for analyzing the protein content of a
mixture. For this experiment, you will be conducting a specific form of PAGE where the proteins remain
in their native states (native PAGE). For comparison, you will conduct another type of PAGE where the
proteins will all be denatured (SDS-PAGE) in another experiment. For both of these experiments, you
will analyze samples from your protein purification of LDH. What do you expect to observe when
comparing the samples after each purification on a PAGE gel? Because the proteins are separated in the
gel, you should expect to see fewer bands as the purification progresses.
There are two reasons that native PAGE provides a stringent test of the purity of that protein. First, the
migration of a native protein through a polyacrylamide matrix is a function of both its charge at the
selected pH and the resistance to its migration produced by the selected percentage of polyacrylamide
in the resolving gel. Each protein responds to the pH and the percentage of acrylamide in its own
characteristic way. As a result, if two proteins happen to have the same electrophoretic mobility at one
LDH Exercise 8: Electrophoresis of Native Proteins on Polyacrylamide
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resolving gel pH and concentration of polyacrylamide, they will have different mobilities at another pH
or at another concentration of polyacrylamide. If only one stained band is observed under two sets of
conditions, (i.e. two different pHs or two different concentrations of polyacrylamide, that band very
likely represents only one protein and the sample can be deemed pure. Second, because the protein
remains in its native conformation during electrophoresis, it retains its biologic activity. Therefore, the
band of protein observed can be assayed for the particular biologic activity being purified, and it can
thereby be demonstrated that the only protein observed on the gel is the protein responsible for that
activity. In the case of LDH, the gel can be stained for all protein with Coomassie brilliant blue (this
chemical is related to the Bradford reagent that binds all proteins) and for dehydrogenase activity to
verify that the only bands of protein observed are those of LDH.
Figure 12 Native PAGE. The gel is placed between and upper and lower buffer chamber. Left panel: the upper stacking gel (pH
6.8) is poured on top of the lower resolving gel (typically pH 8.8). Middle panel: due to the pH of the stacking gel, the proteins
form a sharp band In the stacking gel once voltage is applied. Right panel: once the proteins reach the resolving gel, they begin
to separate from one another based upon their charge, size, and shape. Arrows indicate the distances measured for calculating
𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒃𝒂𝒏𝒅 𝒎𝒊𝒈𝒓𝒂𝒕𝒆𝒅
Rm (relative mobility) = 𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒊𝒐𝒏 𝒇𝒓𝒐𝒏𝒕 𝒎𝒊𝒈𝒓𝒂𝒕𝒆𝒅.
You will be using a technique known as discontinuous gel electrophoresis where two gels are poured.
The lower gel is the resolving gel or separating gel and is typically poured first with a buffer at pH 8.8.
Immediately after pouring the resolving gel, alcohol is layered on top. The alcohol is less dense than the
acrylamide mix and remains on the top. The layer of alcohol serves to make the interface between the
resolving and stacking gels level. Similar to what causes a meniscus when liquids are placed in a
graduated cylinder or serological pipette, capillary action pulls up the liquid level near the left and right
sides of the gel. This uneven surface can result in crooked protein bands. When alcohol is added, it
becomes subject to capillary action instead of the acrylamide solution. The alcohol remains a liquid and
is removed before pouring the upper gel.
The upper gel is the stacking gel with a buffer at pH of 6.8. The protein samples are mixed with a
sample loading buffer and loaded in wells in the stacking gel. This solution contains buffer pH 6.8,
glycerol, and bromophenol blue. The buffer pH is chosen to match the stacking gel pH. Glycerol is a
dense liquid that, when added to the sample, ensures that it will sink into the wells instead of float out
into the upper buffer chamber. Other chemicals that increases a solution’s density can be used in the
place of glycerol (e.g. sucrose, Ficoll, etc.). Bromophenol blue is a pH indicator dye with a pKa ~4.0. The
low pKa ensures that the dye exists in its anionic state so that it will migrate into the gel.
LDH Exercise 8: Electrophoresis of Native Proteins on Polyacrylamide
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When voltage is applied to the system, negatively charged ions, bromophenol blue, and proteins begin
migrating toward the positive electrode. At pH 6.8, glycine is slightly negatively charged and forms a
lagging ion front. Chloride ions (from the Tris-HCl buffer used in the stacking gel) form a leading ion
front and the proteins are “stacked” in the middle, forming a sharp band. Once the proteins reach the
resolving gel, glycine becomes more negatively charged and becomes part of the leading ion front,
allowing the proteins to separate from each other based upon their physical properties (size/molecular
weight, shape, and charge).
The gel is run until the ion front nears the bottom of the gel. Normally you would not be able to see the
ion front since chloride ions are invisible to us. Bromophenol blue, on the other hand, is readily visible
and migrates with the ion front due to its low pKa. The proteins are similarly invisible. We will be using
two staining techniques to locate proteins that have separated in the gel. By far the most common stain
is Coomassie Blue. This dye will bind to all proteins indiscriminately. You will be using a non-toxic
version of this dye called, Simply Blue. The second staining method is an activity stain. In this
experiment, proteins will be maintained in their native states by using nondenaturing buffers and
keeping the gel system cold. This allows you to take advantage of LDH enzymatic activity to locate the
protein in the gel and you will use the same activity stain as was utilized in Exercise 4. In contrast to the
Coomassie Blue staining technique, the activity stain is specific since it only stains the location of LDH.
Armed with this knowledge, can you predict what should be observed if you analyzed your stored
freezer samples of heart LDH on a native PAGE and used the activity stain? What about if you used the
Coomassie Blue stain?
For a single native PAGE, the size/molecular weight, shape, and charge cannot be determined since the
three factors act simultaneously during the separation in the native PAGE. Comparing the migration of a
protein in native PAGE experiments run with different acrylamide concentrations can yield information
about the protein’s molecular weight and relative charge, however.
LDH Exercise 8: Electrophoresis of Native Proteins on Polyacrylamide
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Figure 13 Hedrick-Smith Analysis of Native PAGE. Molecular weights of native proteins can be obtained from Hedrick Smith
analysis. The same set of proteins are run on different percentage native PAGE. The relative mobilites (R m) of each band
(representing each protein) is calculated and the log R m is plotted against the percentage gel. A line is interpolated for each
protein and the slope is calculated. A linear relationship is observed when the slope is plotted against molecular weight.
Although the migration rates of the different proteins in a native PAGE is dependent upon many factors
[the size (molecular weight), shape, and charge], the change in migration rates of each protein observed
in different percentage gels is related to only the protein’s molecular weight. That is, the native gel can
yield information regarding a protein’s molecular weight. The procedure is outlined in Figure 13. For
this experiment, you will use bovine serum albumin (BSA), which has a known molecular weight of
66,000 Da, to create a slope vs. molecular weight standard curve. From this curve you can interpolate
the molecular weight of LDH.
Preliminary Calculations:
You should have already calculated the activity concentration (in mol min-1 mL-1) and mass
concentration (in mg/mL) of the initial supernatant and postcolumn dialysate. Before you come to the
laboratory, refer to the subsection: Preparing dilutions of samples for native PAGE electrophoresis.
Calculate the volume of each extract or dialysate that will contain the appropriate mass of protein or
activity (in mol min-1) for each lane. Write down precisely how you will prepare the dilutions before
you come to the laboratory. You must come prepared to this laboratory with all of your data and
calculations done in advance or you risk not finishing the laboratory on time!
Selected References:
Boyer, R (2006) Characterization of proteins and nucleic acids by electrophoresis. In: Biochemistry
Laboratory: Modern Theory and Techniques, Pearson/Benjamin Cummings, pp 176-186.
LDH Exercise 8: Electrophoresis of Native Proteins on Polyacrylamide
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Hedrick, J. and Smith, A. (1968) Archives of Biochemistry and Biophysics. 126: 155-164 Size and Charge
Isomer Separation and Estimation of Molecular Weights of Proteins by Disc Gel Electrophoresis.
Kyte, J. (1995) Structure in Protein Chemistry, Garland Publishing, New York, pp. 25-38. Dietz, A.A., and
Lubrano, T. (1967) Analytical Biochemistry 20: 246-257.
Ornstein, L. (1964) Annals NY Academy Sci 121: 321-349. Disc electrophoresis I. Background & theory.
Davis, B.J., (1964) Annals N.Y. Academy Sci. 121: 404-427. Disc electrophoresis II. Method & application
to human serum proteins.
Tulchin, N, Ornstein, L and Davis, BJ (1976) Anal Biochem 72:485-490. A microgel system for disc
electrophoresis.
Materials:
In cold room: Bovine Serum Albumin (BSA; 10 mg/mL), 0.25% bromophenol blue, pure LDH from skeletal
muscle and heart tissue (TA may have set out dilutions – check labels); 100 mM pyruvate,
30%Acrylamide:1%bisacrylamide (handle with gloves); ammonium persulfate; Power supplies; 50
mg/mL NAD+; 30% (w/v) sodium lactate; 3 mg/mL phenazine methosulfate; 3 mg/mL nitroblue
tetrazolium
In -20°C freezer: LDH samples; 15 mM NADH,
In lab: 4X Tris-HCl for native stacking gels (pH 6.8); 4X Tris-HCl for native resolving gels (pH8.8); 40 %
sucrose, 0.25% bromophenol blue loading dye; 10 X Tris glycine (pH 8.8); TEMED (in fume hood); 100
mM phosphate buffer; pH 7.5; 500 mM phosphate buffer, pH 7.5; Spec 20; pipets, tips and eppendorf
tubes; glass culture tubes; glass plates and spacers for electrophoresis; 15 well combs; casting stands;
Electrophoresis Chambers; Simply Blue; 20% NaCl; Tupperware and glass containers; plastic tray for
staining; lightbox, scanner and camera; rulers
Safety: Acrylamide is a neurotoxin. Wear gloves and goggles. Pour unpolymerized acrylamide in the
appropriate waste container. Polymerized acrylamide can be disposed of in the trash, but not down the
drain. Phenazine methosulfate and nitroblue tetrazolium may cause skin and eye irritation and must go
in the appropriate waste container. (All three compounds may go in the same waste container – check
labels) Use caution with the electrophoresis chamber to avoid electric shock. Turn off the power supply
and disconnect from the electrophoresis chamber at least 20s before removing lid and touching
samples.
Time Management Guidelines
(60 min) Prepare the stacking gel, samples
(120 min) run gel
(45 min) stain gel
(30 min) image gel
Procedure:
Day 1 – Pour resolving gel and calculate volumes for samples
LDH Exercise 8: Electrophoresis of Native Proteins on Polyacrylamide
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Pour the Resolving Gel
Your TA will assign your group the concentration of acrylamide you will use for your native PAGE.
The concentration will be 6%, 7%, 8%, or 9% acrylamide.
1) Calculate the volume of the 30% acrylamide stock reagent you will need to make 30 mL at the
appropriate percentage.
2) Mix the following in a 15 mL falcon tube by swirling
a. 2.5 mL of 4X Tris HCl buffer for native resolving gel, pH 8.8
b. XX mL of 30% acrylamide, 0.8% bisacrylamide (neurotoxin; calculated from step 1)
c. XX mL of water to make the final volume 10 mL
3) The next step initiates polymerization. Have the gel casting apparatus prepared so that you can
pour the gel before it polymerizes in the Erlenmeyer flask!
a. Refer to the appendix in the lab manual for gel casting. A TA will demonstrate. Make
sure the gel plate sandwich is clamped securely in the gel caster. Insert the appropriate
comb. The interface between the separating and stacking gel should be approximately
1.5-2 cm below the bottom of the sample wells. Estimate and mark where you wish to
fill resolving gel using a Sharpie pen, (Do not use the VWR or Fisher brand ethanol
resistant markers).
b. Add 70 µL of 10% ammonium persulfate and mix tube by inverting
c. Add 8 µL TEMED and mix thoroughly by in verting. Oxygen quenches the
polymerization reaction so do not mix to vigorously.
4) Use a 10 mL pipet to pour the acrylamide mixture between the gel plates allowing it to run
down the side. Avoiding creating bubbles. Fill to the line made (1.5-2 cm below the bottom of
the sample wells) and then gently add a layer of ethanol on top.
a. Check for leaks after a few minutes have passed
5) Allow to polymerize for 20 – 30 min.
a. At times, polymerization of the excess acrylamide in the Erlenmeyer flask will indicate
when the reaction is complete. However, the high surface area to volume ratio of the
liquid in the flask allows oxygen to slow the reaction.
b. The best way to determine whether the reaction is complete is by gently tipping the
casting apparatus. The ethanol will pool at the lower end, revealing solid acrylamide
gel.
6) You can wait to cast the stacking gel next lab period or you can pour it the same day. You will
use a comb to create 15 sample wells in the stacking gel.
a. Store the gel by pouring off the ethanol/unpolymerized acrylamide into the acrylamide
waste.
b. (Skip this step if pouring the stacking gel today). Dilute the 4x (four times the working
concentration) Tris buffer pH 8.8 to 1x (make approximately 4 mL). Add to the top of
the resolving gel. Use tape to seal, label with your name and section, and store upright
in the cold room. (A plastic tube holder/fraction collector rack works well for storing the
entire section’s gels).
c. If wish to get a head start, you can pour the stacking gel (procedure is below). You must
have enough time to wait for it to polymerize, though. Do not store your gel with the
comb since they will be needed for other sections. Take the comb out very carefully
since lane dividers tear easily.
Calculate the amount of sample needed for electrophoresis. Use the table below. Tube 1 is completed
for you.
SHOULD BE COMPLETED BEFORE YOU COME TO CLASS (points)
LDH Exercise 8: Electrophoresis of Native Proteins on Polyacrylamide
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Table 8-2 Sample preparation for Native PAGE
Tube
Protein
Amount
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
25 g
75 g
150 g
75 g
150 g
0.05 U
0.2 U
0.8 U
25 g
125 g
150 g
75 g
0.05 U
0.2 U
0.8 U
Sample
BSA Standard
BSA Standard
Heart crude extract
Purified LDH
Purified LDH
Heart crude extract
Purified LDH
H4 Standard
BSA Standard
BSA Standard
Heart crude extract
Purified LDH
Heart crude extract
Purified LDH
H4 Standard
4x Tris-HCl
6.8 for native
stacking gel
(L)
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
40% sucrose
0.25%
bromophenol
blue (L)
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Protein
(L)
Distilled
water (bring
volume to
50 L) (L)
U = unit = mole/min
1) Standards
a. BSA
i. This will be used as the molecular weight standard for drawing a standard curve
in the Hedrick-Smith analysis (MW of BSA monomer is 66000 Da)
ii. The concentration of BSA you will be given is at a protein concentration of 10
mg/mL. You will need to add an appropriate volume to a tube to obtain the
correct mass of protein to load on the gel.
b. M4 and H4 LDH standards (obtain a 1/10 dilution of the standard from you TA)
i. These will be the same standards that were used in Exercise 4. The amount you
will be adding is in U (units = 1 mole product formed/min).
ii. Use the values of activity concentration that were calculated in Exercise 4 to
determine what volume to add to measure the correct amount of activity.
2) Protein Samples. THIS TABLE SHOULD BE COMPLETED BY THE TIME YOU COME TO CLASS (points)
a. Heart crude extract and Purified LDH
i. The calculations for these samples are analogous to those completed for BSA
and the M4 and H4 LDH standards.
1. You calculated the protein concentrations and the activity
concentrations for these two samples in the protein purification table in
Exercise 7. Use these values to determine what volume you need to
measure.
2. Preparing the samples is not recommended until day 2 since enzyme
activity may decrease upon dilution and storage.
3. If your sample is too dilute, add the maximum volume.
LDH Exercise 8: Electrophoresis of Native Proteins on Polyacrylamide
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a. If you are to make 40 L of sample for the gel and 4 L will be
loading dye and 2 L will be buffer, then you can add a
maximum of 34 L of LDH sample.
Day 2 – Prepare samples, run Native PAGE, and stain
Prepare the 4.5% Native PAGE Stacking gel
1) Take the resolving gel out of cold room and pour off the buffer. You do not need to rinse.
2) Place the gel sandwich with the bag in the casting chamber and obtain the 15 well comb.
3) Mix the following in a 15 mL falcon tube – be sure that the chamber is ready and the
comb is available.
a. 0.75 mL of 30% acrylamide, 0.8% bisacrylamide (neurotoxin)
b. 1.25 mL of 4X Tris HCl buffer the for native stacking gel, pH 6.8
c. 3.0 mL of water
d. 0.050 mL of 0.25% bromophenol blue
i. Adding bromophenol blue to the stacking gel is a nonstandard practice. You will
add it in your gels in this class to assist you with locating the wells they can be
difficult to see.
e. Add 35 µL of 10% ammonium persulfate and mix tube by inverting
f. Add 8 µL TEMED and mix thoroughly
4) Use a pipet to dispense the 4.5% acrylamide mixture between the plates. Avoiding creating
bubbles. Fill to the edge of the glass plate and then carefully insert the comb.
5) Allow to polymerize for 15 - 20 min. If possible, do not remove the comb until it is in the
electrophoresis chamber and submerged in buffer.
a. If you going to run your gel today, prepare your samples
b. If you must remove the comb without the gel being submerged, be careful since the
lane separators tend to break.
i. Pointers for removing the comb:
1. Wiggle the comb while you pull the comb out to allow air to enter the
wells
2. Do not be so concerned about crooked wells. After the comb is
removed, straighten the well dividers with a Hamilton (glass, blunt
needle) syringe.
Prepare the samples for loading
1) Using the table prepared on Day 1, mix all reagents in Eppendorf tubes and store on ice. You
may need to centrifuge briefly to collect the samples at the bottom of the tubes.
Continue preparing for the Native PAGE
6) Make 1X running buffer by diluting the 10X Tris Glycine with DI water.
a. 200 mL for the inner chamber (fits 2 acrylamide gels)
b. 400 mL for the outer chamber of the apparatus (4 gels total), only one group has to make
this
7) With the comb in place, remove the gel sandwich with plates from the casting chamber and use
a razor blade to cut off the plastic bag. The liquid in the bag should not be hazardous if the
acrylamide is polymerized and can be poured down the drain.
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8) Bring the gel and electrophoresis chamber into the cold room. Place the gel sandwich with
spacers and both plates in the electrophoresis chamber with the notched plate facing the inside
and clamp securely. Fill the upper chamber with 1X running buffer and carefully remove the
comb.
9) You may want to rinse the wells, by squirting buffer into the wells with a Pasteur pipet. This
often helps remove unwanted acrylamide strands and makes loading the samples easier. Be
careful not to disturb the sides of the wells, though!
10) Check the chamber for leaks and adjust if necessary.
a. Leaking may cause the gel to run out of buffer before it finishes and you may have to
disassemble the entire setup. Doing so may cause you to lose your samples. Have your
TA check your gel before proceeding. Then fill the lower chamber with 1X running
buffer to the fill line.
11) Place the lid on the apparatus and connect to power supply. Make sure that lid snaps on
securely. Turn the power supply to 200 V, constant voltage, and check the current – it should be
~40 mA per gel. If this is ok, turn off and disconnect.
12) Load samples: Using a P200, pipet 20 µL of each sample into lanes as outlined below. You will
be staining the gel using two different methods (Coomassie and activity stain) and then will
make comparisons between the two halves. To make the comparison easier, load the left half
and right half with the samples in the same order (use the order listed in the Table 8-2).
a. Place a piece of lab tape on the left hand side of the gel, where you will load Tube 1.
13) Close the chamber as before, connect to power supply, and set to 200 V and run for 45 min,
check the dye front, you may need to run it for a bit longer (should be 1 cm from the bottom of
the plate)
14) You may wish to check on the setup periodically to make sure there is enough buffer in the
inner chamber and the system did not accidentally turn off.
15) Turn off the power supply and disconnect chamber. If another group was sharing your power
supply, be sure to turn it back on. Remove the lid and pour the buffer down the drain in the
cold room and then bring the chamber to your lab bench.
Preparing Gel for Staining.
16) Remove the glass plates with your gel from the chamber and set on paper towels on your lab
bench.
17) Prepare two containers as follows (this can be done by one group member while the other is
disassembling the chamber)
a. Simply Blue stain (use a Tupperware with a lid that seals as the container) – put a piece
of tape with your names and section number and fill with DI water.
b. Activity Stain - Mix the following in a 15 mL tube and pour into a plastic container large
enough to hold half of a gel. The container does not need a lid.
i. 7 mL nitroblue tetrazolium (3 mg/mL)
ii. 0.8 mL phenazine methosulfate (3 mg/mL)
iii. 2.0 mL of NAD+ (50 mg/mL)
iv. 1.4 mL of 30% sodium lactate
18) IMPORTANT: Keep in mind the orientation of the gel and remember where lane 1 is located.
Take the spacers out of the glass plates and use a spacer or spatula to gently pry the plates
apart. The gel should stay on one of the plates. If the gel sticks to the top plate and you have to
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flip it over, keep in mind that the gel is also flipped and the lanes are in the reverse order. Then
notch the lower left hand corners of both gels by removing a small portion of the acrylamide.
Gel 1 will be used for the Simply Blue stain; Gel 2 will be used for the activity stain. You can
remove the stacking gel.
Figure 14
19) Staining: You can perform both staining procedures simultaneously since you do not want either
gel half to dry out.
a. Simply Blue – microwave method (Do not leave the gel overnight in Simply Blue without
salt or in DI water alone – the protein can diffuse out). The used reagents can go in the
sink for this staining protocol.
i. Carefully take the gel off of the glass plate and place it in the Tupperware
container with Simply Blue.
1. Mix 135 mL of Simply Blue with 15 mL of 20% NaCl
ii. Microwave for 1min (1 gel) or 1min 30s (2 gels) with Simplye Blue with NaCl.
1. The Simply Blue diffuses into the gel and binds to proteins
iii. Allow to sit for 5 minutes
iv. Destain by decanting dye and rinsing with DI water
1. Rinse until excess dye is no longer seen in the water
2. Destaining is important to remove background stain and improve
visibility of the band for photographing.
v. Add fresh water and heat in microwave for 3 minutes.
1. This will be hot! Handle with care.
vi. Add fresh water and repeat the microwave cycle (3 minutes)
vii. Photograph (instructions below) and measure the migration of bands relative to
the dye front. You do not need to diagram this in lab your lab notebook if the
photo is clear.
b. Activity Stain – all liquid waste goes into the appropriate waste container
i. Cut a piece of Whatman paper the same size as the gel half you set aside for
activity stain.
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ii. Add the Whatman paper to the plastic tray with staining solution and soak until
damp.
iii. Place the Whatman paper on top of gel the on the glass plate. Place Saran wrap
over the Whatman paper and gel and flip over. Watch for the appearance of
bands.
1. The substrates and staining reagents diffuse out of the Whatman paper
into the gel.
2. When bands are visible, but before they blend together, remove the
Whatman paper and Saran wrap and photograph the gel (instructions
below).
3. Draw a diagram of the gel in your lab notebook indicating all visible
bands. Measure the distance the bands traveled relative to the dye
front.
20) Posting the data
a. This lab requires pooling the data from the section. You must make your data available
to your classmates before you leave.
21) Clean up
a. Wash out the gel casting chamber and electrophoresis chamber with DI water and leave
them to dry on your benchtop
b. Discard any excess activity stain in the appropriate waste container.
Capture Image:
1) Place the two gel pieces on a glass plate, align the edges, and place on light box under the
camera.
2) Lay a clear-plastic ruler alongside standards lane. Attempt to mark 0 cm at the top of the
resolving gel.
3) Kodak MI program: set for “white light, transillumination”.
4) Adjust zoom and focus on camera lens.
5) Optimize contrast by adjusting exposure time and/or the aperture on camera lens.
6) Capture image.
Questions to think about:
1. Explain how the stacking gel concentrates the protein into thin bands. What is different about the
way a protein is able to move in the stacking gel compared to the resolving gel?
2. What considerations should be made when determining the percentage acrylamide used in the
resolving gel?
3. How does the Simply Blue stain give discernible bands on a destained gel?
4. The apparatus is designed so that the (red) terminal connected to the (+) port on the power supply
connects with the electrode in the bottom buffer tank. This is the anode. How does the result of the
Cellulose Acetate exercise assure us that the LDH in these samples will migrate towards the anode?
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Exercise 9 SDS-PAGE of the Initial Supernatant and Purified LDH
Key Principles:
Determination of the molecular weight (MW) of a polypeptide; Denaturation of protein by sodium
dodecyl sulfate (SDS); Mass to charge ratio; Sieving effect on migration of a complex between dodecyl
sulfate and a polypeptide; Polyacrylamide gel electrophoresis (PAGE).
Description of Procedure:
Native gels give complicated results since the migration of the proteins in the gel is dependent on size,
shape, and charge. A fair amount of consideration must be given to determine the appropriate buffer,
pH, and theoretical charge on the protein of interest to ensure that it migrates into the gel and not the
opposite direction. SDS-PAGE is an alternative and more standard method for analyzing a protein
mixture. Using this method, the experiment is simplified since the proteins migrate based upon their
molecular weight and the same buffer can be always be used.
In the presence of the strong detergent, sodium dodecyl sulfate (SDS), multisubunit proteins are
dissociated into individual unfolded polypeptide chains. Dodecyl sulfate anions bind to most proteins at
a constant ratio of 0.54 anions per amino acid. This causes all proteins to possess the same charge to
mass ratio. The negative charge of the sulfate groups confers a net negative charge to the protein,
masking its intrinsic charge. The negative charge also serves to unfold or denature proteins through
electrostatic repulsion.
SDS is typically added to the gel and to the sample loading buffer. In addition to SDS, mercaptoethanol (ME, or 2ME) is frequently added to the sample loading buffer to help denature
proteins. ME is a reducing agent that reduces cystines (disulfide bonded cystienes). When ME is
added, the experiment is often referred to as a reducing SDS-PAGE. Sometimes protein tertiary or
quaternary structure is stabilized by disulfide bonds and analysis without ME is also performed for
comparison. SDS-PAGE without reducing agent is a nonreducing SDS-PAGE.
Denaturing results in the proteins taking on a more extended conformation and having the same shape.
Under these circumstances, all proteins have comparable charge to mass ratios and similar extended
shapes. The only basis for separation that remains is the sieving effect where large proteins migrate
slower than small proteins. Since size is proportional to molecular weight, the electrophoretic mobility
of all proteins in SDS PAGE becomes dependent on the molecular weight of the polypeptide. The
relationship between migration and MW is not linear, but rather semi-logarithmic (the relative mobility
is directly proportional to the log MW).
Preliminary Calculations:
You should have already determined the concentration of protein (in mg/mL) for the bovine heart initial
supernatant, precolumn dialysate, flow through, and postcolumn (purified LDH) dialysate in Exercise 7.
Refer to the subsection of this exercise on Preparing dilutions of samples for SDS gel electrophoresis.
Before you come to the laboratory, calculate the amount of each extract or dialysate needed to obtain
the mass of protein indicated in the table. Following the directions, write down how you will prepare the
dilutions before you come to the lab. You must come prepared to this laboratory with all of your data
LDH Exercise 9: SDS-PAGE of the Initial Supernatant and Purified LDH
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and calculations done in advance or you will not finish the laboratory on time. Organization will be
critical.
Selected References:
Boyer, R (2006) Characterization of proteins and nucleic acids by electrophoresis. In: Biochemistry
Laboratory: Modern Theory and Techniques, Pearson/Benjamin Cummings, pp 176-186.
Kyte, J. (1995) Structure in Protein Chemistry, Garland Publishing, New York, pp. 291-300. Laemmli, U.K.
(1970) Nature 227: 680-685.
Scopes, R.K. (1994) Protein Purification, 3rd ed, Springer-Verlag, New York, pp. 250-258, 293-298, 302303.
Weber, K., and Osborne, M. (1969) J. Biol. Chem. 244:4406-4412.
Materials:
In cold room: 0.25% bromophenol blue, water at ~75oC, LDH samples; MW standards; 30% Acrylamide:
0.8% bisacrylamide (handle with gloves); 10% ammonium persulfate (made fresh)
In -20°C freezer: LDH samples
In lab: 4X Tris-HCl with SDS for stacking gels (pH 6.8); 4X Tris-HCl with SDS resolving gels (pH8.8); 80 %
glycerol, 0.25% bromophenol blue loading dye; 10% β-mercaptoethanol (in fume hood), 50 mM sodium
phosphate, pH 7.2; 10% SDS; 10 X Tris -glycine with SDS (pH 8.8); TEMED (in fume hood); 100 mM
phosphate buffer; pH 7.5; pipets, tips and eppendorf tubes; glass plates and spacers for electrophoresis;
10 or 15 well combs; ethanol for gel polymerization; casting stands; boiling water bath; Electrophoresis
Chambers; Power Supplies; Simply Blue; 20% NaCl; Tupperware and glass containers; lightbox, scanner
and camera; rulers
Safety: Acrylamide is a neurotoxin. Wear gloves and goggles. Pour unpolymerized in the appropriate
waste container. Polymerized acrylamide can be disposed of in the trash – not down the drain. Use
caution with the electrophoresis chamber to avoid electric shock. Turn off the power supply and
disconnect it from the electrophoresis chamber for at least 20s before removing the lid and touching
samples.
Time Management Guidelines
(60 min) Prepare the stacking gel, samples
(120 min) run gel
(45 min) stain gel
(30 min) image gel
Procedure:
Pour the 12.5% SDS Resolving gel.
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The procedure is the same as for the nondenaturing gel, however the gel formulation is different. Be
sure the gel plate sandwich is clamped securely in the gel caster and the fill level is marked using the
appropriate comb before you make the acrylamide mixture.
1) Mix the following in a 15 mL falcon tube by swirling
a. 4.2 mL of 30% acrylamide, 0.8% bisacrylamide (neurotoxin)
b. 2.5 mL of 4X Tris HCl buffer with SDS for resolving gel, pH 8.8
c. 3.3 mL of water
d. Add 70 µL of 10% ammonium persulfate and mix
e. Add 8 µL TEMED and mix – This starts the polymerization so move on quickly to next
step.
2) Use a 10 mL pipet to pour the acrylamide mixture between the gel plates allowing it to run
down the side. Avoiding creating bubbles. Fill to the line made using the comb and then gently
squirt ethanol on top.
3) Allow to polymerize for 20 – 30 min.
a. At times, polymerization of the excess acrylamide in the falcon tube will indicate
when the reaction is complete.
b. The best way to determine whether the reaction is complete is by gently tipping the
casting apparatus. The ethanol will be pool at the lower end, revealing solid acrylamide
gel.
4) Decant the ethanol and rinse the plates a few times with distilled water.
5) If you pour the gel on the same day you are using it, you will not need to complete this step –
continue on.
a. If you are storing the gel overnight, fill the space above the gel with 1X Tris-Cl, pH 8.8
(you will need to dilute some of the 4X stock). Remove the gel sandwich still in the
plastic bag from the casting stand and seal with tape. Label with your name and section
number and store the gel in the cold room.
Day 2: Prepare LDH samples and run the gel. One partner can focus on preparing the samples while the
other can focus on preparing the gel.
Pour the 4.5% SDS Stacking Gel
6) If the gel was stored, take the resolving gel out of the cold room and pour off buffer. You do not
need to rinse.
7) Place the gel sandwich with the bag in the casting chamber and obtain a 10 well comb.
8) Mix the following in a 15 mL falcon tube – be sure that the chamber is ready and comb is
available.
a. 0.75 mL of 30% acrylamide, 0.8% bisacrylamide (neurotoxin)
b. 1.25 mL of 4X Tris HCl buffer with SDS for stacking gel, pH 6.8
c. 3.0 mL of water
d. 0.050 mL of 0.25% bromophenol blue
i. Adding bromophenol blue to the stacking gel is a nonstandard practice. You will
add it in your gels in this class to assist you with locating the wells they can be
difficult to see.
e. Add 35 µL of 10% ammonium persulfate and mix
f. Add 8 µL TEMED and mix – This gel will polymerize much more quickly than the
first.
9) Use a 10 mL pipet to pour the 4.5% acrylamide mixture between the plates allowing it to run
down the side and avoiding bubbles. Fill to the edge of the glass plate and then insert a 10 well
comb carefully.
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10) Allow to polymerize for 15 - 20 min.
Electrophoresis and Staining
11) Make of 1X running buffer by diluting the 10X Tris Glycine +SDS with DI water.
i. 200 mL for the inner chamber (fits 2 acrylamide gels)
ii. 400 mL for the outer chamber of the apparatus (4 gels max), only one group has to make this
12) With the comb in place, remove the gel sandwich with plates from the casting chamber and use
a razor blade to cut off the plastic bag. The liquid in the bag should not be hazardous if the
acrylamide is polymerized and can be poured down the drain. At this point you can mark your
wells with a sharpie – on the square plate (be sure to clean this off using ethanol when you are
finished).
13) This gel will be run at room temperature. Set up the electrophoresis chamber on your lab
bench. Place the gel sandwich in the electrophoresis chamber with the notched plate facing the
inside and clamp securely. Fill the upper chamber with 1X Running buffer +SDS and carefully
remove the comb. You may want to rinse the wells, by squirting buffer into the wells with a
Pasteur pipet.
14) Check the chamber for leaks and adjust if necessary. If you load the samples and there are leaks
in the apparatus, you may lose your samples. Then fill the lower chamber with 1X running
buffer to the fill line.
15) Place the lid on the apparatus and connect to power supply. Make sure that lid snaps on
securely. Turn the power supply to 200 V, constant voltage, and check the current – should be
~40 mA. If this is ok, turn off and disconnect.
Prepare dilutions of samples for SDS gel electrophoresis
16) Using the table below, mix all reagents in Eppendorf tubes.
17) Heat Eppendof tubes for 5 min in the 90 degrees C heat block
18) Let cool and centrifuge for 5s to collect the samples at the bottom of the tubes.
Sample loading table (Your volumes will be different)
SHOULD BE COMPLETED BEFORE YOU COME TO CLASS (points)
Tube
1
2
3
4
5
6
7
8
9
10
Protein Sample
Amount
(g)
4
4
150
150
200
200
200
200
200
Standard*
H4
M4
Heart Crude Extract
Precolumn dialysate
Flow Through
0.05 M elution
0.25 M elution
0.50 M elution
1.00 M elution
10% mercaptoethanol
50 mM sodium
phosphate, pH 7.2
(L)
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
80% glycerol 10%
0.25%
SDS
bromophenol (L)
blue (L)
-
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
-
1
1
1
1
1
1
1
1
1
Protein
(L)
5
Distilled
water
(bring
volume to
50L) (L)
-
*NOTE: There are two types of standards, one is a pre-stained and ONLY used for Western Blots and much more expensive.
19) Load samples: Using P200, pipet 20 µL of each sample into the lanes. If your protein is not
concentrated enough, then add as much volume of protein as you can (add 0 L of water).
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20) Close the chamber as before, connect to power supply, and set to 200 V and run for 45 min.
These gels tend to run faster and hotter than the native PAGE, may need to run for longer for
the dye front to reach 1cm above the bottom plate.
a. Check periodically to ensure there is enough buffer in the inner chamber and that the
power supply is still running.
21) Turn off the power supply and disconnect the chamber. Remove the lid and pour the buffer
down the drain and then bring the chamber to your lab bench.
22) Prepare the gel for staining using Simply Blue. Refer the section in the native PAGE experiment.
23) Remove the glass plates with your gel from the chamber and set them on paper towels on your
lab bench – notched plate on the bottom.
24) Take spacers out of glass plates and use a spacer spatula to gently separate plates.
a. Simply Blue – microwave method (Do not leave the gel overnight in Simply Blue without
salt or in DI water alone – the protein can diffuse out). The used reagents can go in the
sink for this staining protocol.
i. Carefully take the gel off of the glass plate and place it in the Tupperware
container with DI water.
1. Mix 135 mL of Simply Blue with 15 mL of 20% NaCl
ii. Microwave for 1min (1 gel) or 1min 30s (2 gels) with Simply Blue with NaCl.
1. The Simply Blue diffuses into the gel and binds to proteins.
iii. Allow to sit for 5 minutes
iv. Destain by decanting dye and rinsing with DI water
1. Rinse until excess dye is no longer seen in the water
2. Destaining is important to remove background stain and improve
visibility of the band for photographing.
v. Add fresh water and heat in microwave for 3 minutes.
1. This will be hot! Handle with care.
vi. Add fresh water and repeat the microwave cycle (3 minutes)
iii. Photograph and measure the migration of bands for the standards and the LDH
in the purified sample. Do not attempt to measure all of the bands (especially in
the unpurified sample). You do not need to diagram this in lab your lab
notebook if the photo is clear.
25) Clean up
a. Clean off plates with soap, water and then rinse with DI water and ethanol. Wash off all
Sharpie marks with ethanol before returning to tub.
b. Wash out the gel casting chamber and electrophoresis chamber with DI water and leave
to dry on your benchtop
Handling the Data:
LDH Exercise 9: SDS-PAGE of the Initial Supernatant and Purified LDH
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1) A semilog plot for the protein migration of the standards must be created by one of the
following three methods:
a. Use single-cycle semilog paper with the MWs of the standards on the y-axis (log scale)
and distance migrated (in mm) on the x-axis (linear scale).
b. Calculate the log10(MW) for each standard. Using conventional graph paper, plot these
values on the y axis as a function of distance migrated (in mm) on the x axis.
c. Use MS-Excel graphing log10(MW) on the y-axis and distance (mm) on the x-axis
2) Determine the MW of a polypeptide of LDH by interpolation on the semilog plot. Keep in mind
that if you use the equation from the trend line using Excel, the y value is the log 10(MW) and
must be converted to the MW.
3) Qualitatively compare the degree of purification between the initial supernatant, the precolumn
sample, and the postcolumn sample.
Questions to think about:
1.
What are two functions of the SDS in this type of electrophoresis?
2.
Why is 2-mercaptoethanol added to the samples?
3.
Explain the order in which the components are added to the sample of protein before gel
loading.
4.
What are some drawbacks to using this method to determine the molecular mass of a
polypeptide? What factors might affect its accuracy? Why might membrane proteins migrate
anomalously fast on SDS-PAGE?
5.
How is this technique different from cellulose acetate electrophoresis?
6.
Suggest a method of verifying that the band that you believe to be LDH is indeed LDH.
7.
If you were separating polypeptides that had lengths in the range of 100 to 300 amino acids,
would you use a higher or a lower concentration of acrylamide? Why?
Acknowledgement: We thank Keith Wood for detailed and helpful descriptions of the assembly and
preparation of the SDS-PAGE gels described in the publication of Laemmli.
LDH Exercise 9: SDS-PAGE of the Initial Supernatant and Purified LDH
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Exercise 10 Western Blot
The term “Western Blot” is not very descriptive of the actual technique. The name alludes to another
technique known as the Southern Blot, named after its developer (Edwin Southern) where DNA is
separated electrophoretically and then detected with a DNA probe that can bind specifically to a DNA
sequence of interest by complementary base pairing. The Western Blotting technique is analogous to
Southern Blotting except rather than using a DNA probe to locate a DNA of interest following
electrophoresis, a protein probe is used to locate a protein of interest.
Understanding how DNA can be used as a probe is fairly straightforward if you understand the principle
of complementary base pairing. But how can you use a protein as a probe to specifically detect a
protein of interest? The key to this question comes from a topic in Immunology. We, like virtually all
other animals, are constantly under attack by microorganisms that would like nothing more than to
break down our complex molecules to obtain energy that is needed to keep them alive. As evidence,
consider how quickly meats spoil if left out at room temperature. To deal with these pathogens, we
have developed an immune system that that recognizes and destroys invading organisms. Without our
immune system, we would succumb to the microbes just as the meat.
There are two branches of the immune system, adaptive and innate. For our purposes, we will focus on
an aspect of the adaptive immune system. When an invading pathogen enters our system, a certain set
of immune cells that circulate in the blood are activated. These cells are known as B-cells, though the
“B” stands for bone marrow, where they mature. These cells have a special protein on their surface
called a B-cell receptor, and each B-cell has a unique variant of the B-cell receptor. These cells are
prescreened such that any B-cell with a receptor that recognizes a protein found naturally in the host
organism is killed (clonal deletion). Thus, only B-cells that recognize foreign proteins circulate in the
blood.
When a B-cell receptor recognizes a foreign protein, otherwise known as an antigen, the B-cell receives
signals to begin dividing and secreting a version of the B-cell receptor. This secreted B-cell receptor is an
antibody. Thus an antibody is a protein that is capable of specifically recognizing an antigen. Their
specificity for their antigen is what makes them useful as a probe in Western Blots. An antibody can be
raised to target virtually any protein of interest. This is accomplished by taking the protein of interest
and injecting it into a different species that possesses an adaptive immune response. For example, you
will use an antibody that was made by intravenously administering bovine LDH to rabbits. The rabbit
immune system generated antibodies against bovine LDH which were then collected from the rabbit’s
blood. Note that this process wouldn’t work by injecting bovine LDH into cows because the B-cells that
could have recognized the bovine form of LDH are killed. Interestingly, faulty clonal deletion will result
in production of antibodies that attack the host and is a possible mechanism for autoimmune disorders.
Now that you understand that an antibody has a specific affinity for its antigen and can be used as a
probe to detect its antigen in mixture of proteins, we shall examine the Western Blotting technique.
The first step of Western Blotting is exactly the same as SDS-PAGE. Protein samples are separated
electrophoretically on an SDS polyacrylamide gel. Following separation, the proteins are
electrophoretically transferred out of the gel onto a membrane made of PVDF (polyvinylidine fluoride)
or nitrocellulose which readily adsorbs proteins (see Figure 15). This transfer is necessary since the
antibody probe is a large protein (~150000 Da) that cannot diffuse into the gel to access the antigen.
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Figure 15 Western Blot Transfer. The initial step of the Western Blot the same as SDS-PAGE. Subsequently, the proteins are
transferred to a membrane that readily adsorbs proteins (PVDF or nitrocellulose) by stacking the membrane and the gel and
applying a current across the stack.
Once the proteins have been adsorbed to the surface of the membrane, the antibody probe can readily
bind specifically to the antigen. However, the membrane has an inherent affinity for all proteins so if
the antibody is added at this point, it will also bind to the membrane nonspecifically. To prevent
nonspecific antibody binding, the membrane is blocked. Common block agents are random proteins
such as bovine serum albumin (BSA) or proteins in rehydrated dry milk. Other blocking agents include
weak detergents like Tween-20, which is what you will use in this experiment.
After the blocking step, the antibody probe is incubated with the blocked membrane at which time the
antibody binds to its antigen on the membrane surface. This antibody is the first antibody that is added
so it is often considered the primary antibody. You will be using an antibody that was raised in rabbits
that is directed against bovine LDH. The shorthand way of saying it is “rabbit anti-bovine LDH antibody”
(sometimes written as rabbit -bovineLDH).
Remember that proteins are invisible so you will need some way to develop the Western Blot. For the
native gel, you used an activity stain where you took advantage of the LDH activity. For the Western
Blot, a secondary antibody is used to localize an enzyme to the site of the primary antibody. For this
experiment, the secondary antibody was made by injecting purified rabbit antibodies into a goat. The
goat’s immune system responded by generating anti-rabbit antibodies. These goat antibodies were
purified and then chemically conjugated to biotin, vitamin B7. The protein, avidin, is known to have a
high affinity for biotin. You will add a version of avidin that has been chemically conjugated to an
enzyme, horse radish peroxidase (HRP). In the end, HRP will be co-localized with the protein of interest.
The enzymatic activity of HRP can be used to develop the blot with an activity stain (see Figure 16).
Figure 16 Schematic for developing a Western Blot. Black Y represents primary antibody. Grey Y represents secondary
antibody. HRP is horse radish peroxidase. DAB (3,3'-diaminobenzidine tetrahydrochloride) acts as a substrate for HRP.
As you can see, Western Blots are considerably more complicated than SDS-PAGE. So why would a
researcher resort to Western Blot experiments? One reason is that Western Blots are far more sensitive
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than Coomassie stained SDS-PAGE. To be capable of visualizing a protein band by Coomassie stain, one
typically needs about one microgram. To observe the same band in a Western Blot, only a few
nanograms are needed. The greater sensitivity of the Western Blot technique is probably due to the
high affinity antibodies typically have for their antigen. Also, there is usually a signal increase with each
reagent that is added. In this experiment, multiple secondary antibodies can bind to the primary
antibody (shown in Figure 16).
In addition to being more sensitive, Western Blots analyses are more specific. In a Coomassie stained
SDS-PAGE, all proteins are visualized. If a protein sample being analyzed is not pure (e.g. if it is a tissue
crude extract), the gel would show many bands and it might be impossible to determine whether or not
the protein of interest is present. Due to the specificity of the antibody probe that is used in a Western
Blot, a researcher can detect the presence of a protein of interest even in a complicated mixture of
proteins.
Materials:
In cold room: LDH samples; biotinylated MW standards; 30% Acrylamide:0.8% bisacrylamide (handle
with gloves); 10% ammonium persulfate;
In -20°C freezer: LDH sample; primary and secondary antibodies; diaminobenzidine solution;
In lab: 4X Tris-HCl with SDS for stacking gels (pH 6.8); 4X Tris-HCl with SDS separating gels (pH 8.8); 80 %
glycerol, 0.25% bromophenol blue loading dye; 10% β-mercaptoethanol (in fume hood), 50 mM sodium
phosphate, pH 7.2; 10% SDS; 10 X Tris glycine with SDS (pH 8.8); TEMED (in fume hood); 100 mM
phosphate buffer; pH 7.5; pipets, tips and eppendorf tubes; glass plates and spacers for electrophoresis;
15 well combs; ethanol for gel polymerization; casting stands; boiling water bath; Electrophoresis
Chambers; Power Supplies; Transfer buffer (Tris-Cl pH 8.8, 20% Methanol (degassed for several hours));
nitrocellulose, Whatman paper, transfer chamber; PBS (10 mM Sodium phosphate, pH 7.5, 150 mM
NaCl, TPBS (0.05% v/v Tween-20 in PBS), NiCl2, Tupperware and glass containers; lightbox, scanner and
camera; rulers
Safety: Acrylamide is a neurotoxin. Wear gloves and goggles. Pour unpolymerized in appropriate waste
container Polymerized acrylamide can be disposed of in the trash – not down the drain.
Diaminobenzidine is a carcinogen – dispose of in the assigned waste container. Use caution with the
electrophoresis chamber to avoid electric shock. Turn off the power supply and disconnect the
apparatus from the electrophoresis chamber at least 20s before removing the lid and touching the
samples.
Procedure: Day 1
1) Pour a 12.5% SDS-PAGE as done in Exercise 9.
2) Prepare the samples for loading. One partner can set this up while the other prepares the
stacking gel.
3) Using the table below, mix all reagents in Eppendorf tubes and store on ice. You can centrifuge
briefly to collect the samples at the bottom of the tubes.
Table 1: Samples for SDS-PAGE
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Table 0-3 Western Blot samples. Lanes 1-5 will be developed using the rabbit -LDH primary antibody. Lanes 6-10 will be
developd using a rabbit -BSA primary antibody.
Tube Protein
Amount
(g)
Sample (g)
1
2
3
0.5
25
0.1
4
1
5
6
7
8
9
10
10
10
25
1
0.1
1
H4 standard
Heart Crude Extract
Most pure Cib. Blue
elution
Most pure Cib. Blue
elution
Marker
Marker
Heart Crude Extract
Purified LDH
BSA
BSA
10% mercaptoeth
anol 50 mM
sodium
phosphate,
pH 7.2 (L)
4
4
4
80% glycerol
0.25%
bromophenol
blue (L)
10%
SDS
8
8
8
1.6
1.6
1.6
4
8
1.6
4
4
4
4
8
8
8
8
1.6
1.6
1.6
1.6
Protein
(L)
5
5
Distilled water
(bring volume to
40 L) (L)
-
26) Heat Eppendof tubes for 5 min in the 90 degrees C heat block
4) Let cool and centrifuge for 5 sec to collect the liquid at the bottom of the tube
5) Run electrophoresis as done in Exercise 9. Stop gel when bromophenol blue is ~ 1 cm from
bottom.
a. You may wish to place a piece of tape at the top of one of your glass plates to mark the
location of lane 1.
Prepare for the transfer onto nitrocellulose. Handle nitrocellulose with gloves and do not lay it on the
bench top. Remember that it sticks to proteins so it may bind to those on your hand and any found on
your benchtop.
6) While the gel is running, obtain a piece of nitrocellulose slightly larger than the size of your gel.
You can use a glass plate to estimate the size of your gel. The membrane will be cut in half so
mark with your initials on the both left and right edges and cut a small piece from the lower left
hand corner to help you keep track of the orientation. Float the nitrocellulose sheet on distilled
water until wet with no spots.
7) Remove the glass plates with your gel from the chamber and set it on paper towels on your lab
bench – notched plate on the bottom. Take note of the location of lane 1 then take the spacers
out of the glass plates and use a spacer spatula to gently separate plates. The gel should stay on
one of the plates.
8) Cut a small piece at the lower left hand corner under lane 1.
9) Lay the gel on top of the nitrocellulose, smoothing to remove bubbles.
10) Hydrate two sheets of Whatman paper and assemble a Western Blot “Sandwich” as depicted in
Figure 17.
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Figure 17 Western Blot “Sandwich”
11) Place the “Sandwich” between two pieces of Scotch Brite pads as instructed by your TA.
a. The transfer apparatus has two pieces of plastic that will enclose the sandwich. The red
or white piece will be directed toward the (+) electrode. The black piece will be placed
closest to the (-) electrode. Be sure the nitrocellulose is oriented closest to the
white/red plastic.
12) Fill the transfer chamber with degassed 1XTris Glycine, 20% methanol (transfer buffer; your TA
may do this for you). Slide the cassette into the chamber (checking that orientation is correct).
13) When all students have loaded their blots, run for 16 – 22 hr at 100 mA per transfer chamber,
constant current (usually ~34 V).
14) Turn off the power supply. Disconnect the electrodes and remove the cassettes. Carefully open
the cassettes and take out the gel/Whatman/nitrocellulose sandwich.
15) Place the sandwich on a glass plate or in a glass dish and remove the Whatman paper, but not
nitrocellulose.
16) Use a razor blade to cut the nitrocellulose at same point as gel, cutting carefully to avoid losing
data.
17) Rinse in DI water and place in ziplock sandwich bag with TPBS buffer to block. Label ziplock bag
with tape and store in cold room until next lab period. Try to ensure that the blocking buffer
contacts all of the membrane to promote efficient blocking (i.e. try to remove bubbles and air)
Immunostaining
Day 2
1) Pour off TPBS and move each half of the blot to its own small plastic container. Label the
container with your initials and the antibody used
2) Add 20 mL of primary antibody in TPBS – check in cold room for the dilution to use.
a. Incubate for 1 hr at RT with gently shaking.
b. The membrane with lanes 1-5 should receive the -LDH antibody. The membrane with
lanes 6-10 should receive the -BSA antibody.
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3) Pour the primary antibody solution out and rinse 3 times with TPBS by adding TPBS and
incubating with periodic shaking for 5 min for each rinse.
a. If it is available, you can use the shaker.
4) Add 20 mL of biotinylated secondary antibody diluted 1:1000 in TPBS (or as directed).
a. Incubate for 30 min with gentle shaking.
5) Pour out secondary antibody solution and rinse 3 times with TPBS by adding TPBS and
incubating with periodic shaking for 5 min for each rinse.
a. If it is available, you can use the shaker.
6) Add 20 mL of avidin/horseradish peroxidase conjugate diluted 1:7000 in TPBS.
a. Incubate 30 min with gentle shaking.
7) Pour out the avidin/HRP conjugate solution and rinse 3 times with TPBS by adding TPBS and
incubating with periodic shaking for 5 min for each rinse.
a. If it is available, you can use the shaker.
8) Mix the staining solution just prior to use:
a. 1.0 mL of DAB, in 0.5 M Tris, pH 7.2
b. 9 mL of distilled water
c. 50 µL of 8% NiCl2
d. 7.5 µL of 30% H2O2
9) Place the blot in staining solution and watch for distinct bands of red/brown color.
10) Stop the reaction by washing with DI water.
11) Dispose of the dye in the appropriate chemical waste.
12) Photograph blots and record in lab notebook. Try to align both of the membranes with each
other when taking the picture. Again, the signal will decay as the blots dry. Measure the
migration of MW stds, BSA, LDH and any other bands that cross react with the antibody. You
may want to draw a picture in your lab manual.
13) Be sure to obtain the MW information for the biotinylated standards.
Clean up
14) Clean off gel plates using soap and water, rinse well. Use ethanol or acetone to remove Sharpie
marks.
15) Wash out the gel casting chamber and electrophoresis chamber with DI water and leave to dry
on your benchtop
Handling the data
The molecular weight for the protein bands can be calculated as done with the SDS-PAGE experiment.
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LuxG Section II: Expression, purification, and characterization
Introduction
Flavin Reductases, Bioluminescence, and the lux Operon
LuxG
Bioluminescence occurs in a variety of organisms; e.g. firefly, marine invertebrates, and bacteria. Many
luminescent bacteria exist in symbiosis with fish. These bacteria proliferate within certain tissues,
making the fish appear luminescent. The reaction that generates light energy is catalyzed by the
luciferase enzyme in all these organisms. However, the substrates of the reaction vary quite a bit. In
the luminescent marine bacteria, Photobacterium leiognathi and Photobacterium fischeri (previously
known as Vibrio fischeri), one of the substrates is the reduced form of flavin mononucleotide (FMNH -;
Hosseinkhani et al. 2005). Most textbooks show the reduced form as FMNH 2, however at physiological
pH it is expected to be ionized to FMNH- (Song et al. 2007).
Figure 18 Suggested reaction mechanism of the LuxG flavin redutase. Nijvipakul, S et al.
(2008) J Bacteriology 190:1531-1538.
The light-generating reaction involves oxidation of FMNH- to FMN. For sustained luminescence, FMN
must be reduced back to FMNH-. Such a reaction is usually catalyzed by a flavin reductase enzyme
(Ingelman et al. 1999). In P. fischeri and P. leiognathi the genes that encode the luciferase enzyme and
the flavin reductase are part of an operon, or cluster of genes that are controlled by a single promoter.
This is one method cells use to regulate relative gene expression since all genes in the operon will be
transcribed at the same rate. The lux operon contains luxA and luxB which express proteins that
associate with one another to form an -heterodimer and active luciferase enzyme (Winfrey et al.
1997). This operon also includes luxG that codes for a flavin reductase.
The enzymatic assay you will conduct to detect LuxG reductase activity is similar to the LDH assay. Using
FMN and NADH as substrates, NADH will become oxidized as FMN is reduced, and the reaction can be
monitored by a decrease in A340 with a spectrophotometer.
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Figure 19 Predicted secondary structure of the LuxG protein from P. leiognathi. -helices (rectangles) and -strands (arrows)
correspond to those in the experimentally-determined structure of the Fre enzyme from E. coli. Labeling: "F" structures form the
flavin-binding domain, "N" form the NADH-binding domain. Top sequence is that determined for LuxG by the Chaiyen lab. Bottom
sequence was previously submitted to a database (entry: LuxG_AAA25621). Nijvipakul, S et al. (2008) J Bacteriology 190: 1531–1538.
Heterologous Expression
Years ago, scientists were interested in what we may consider basic and perhaps simplistic questions.
They considered questions such as, “How do enzymes catalyze reactions?” and, “What are the
mechanisms of enzymatic inhibition?” Through their work, we have come to better understand
enzymes and we can go over the experiments that they conducted during the course of several years in
one 10 week or 5 week biochemistry class. Like any sensible scientist back then, they focused on
enzymes such as LDH that have readily detectable activities and are abundant in tissues. Biochemists of
today are more focused on proteins related to healthcare which are often expressed at very low levels
and in specific tissues. Obtaining enough protein for study can prove challenging. The majority of
proteins that are studied these days are produced using a heterologous expression system, meaning
that the gene for the protein of interest is placed in cells that normally do not synthesize the protein.
Once the gene is introduced, the cells can begin expressing the protein of interest.
Many of the initial steps necessary for heterologous expression will have been completed for you and
are covered in CHEM 109, which focuses on recombinant DNA technology. Some of the main points will
be briefly covered here so that you will understand the basics of what you are working with. The gene
for luxG was placed inside of a DNA construct known as an expression vector or expression plasmid.
Plasmids are circular strands of DNA that are sometimes found in bacteria. In nature, they can contain
genes that are useful to bacteria, such as antibiotic resistance genes, and are often cited as a reason for
the emergence of multidrug resistance in bacteria.
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Expression plasmids can be constructed, but
many of them are purchased through biotech
companies. They are sold without the gene of
interest incorporated so the researcher must
ligate the gene into the expression plasmid using
molecular biology techniques. A LuxG
expression plasmid construct has been provided
by Dr. P. Chaiyen (Mahidol University, Thailand)
for use in this course. A schematic of the vector
is shown in Figure 20. Once the gene for the
protein of interest is incorporated into the
expression plasmid, it is introduced into E. coli
using a technique called transformation. The
culture of transformed bacteria is grown, and the
expression plasmid is propagated throughout the
bacteria in the culture.
Figure 20 Schemiatic of the LuxG expression plasmid.
As observed in Figure 20, there are a number of
aspects that are important for the expression
vector. The origin of replication and kanamycin resistance gene are important for the maintenance of
plasmid. The origin of replication (ori) is a sequence of DNA that signals the replication of the plasmid
before each cell division. Without the origin of replication, the number of bacteria in the culture would
increase as the culture grows, but the number of expression plasmids would not. After only a few cell
divisions, most of the cells in the culture would be left without the expression plasmid. This situation
would cause the overall expression of LuxG to be poor.
The kanamycin resistance gene allows selection of only those E. coli cells that possess the expression
plasmid. Kanamycin is an antibiotic that kills bacteria by interfering with protein translation. Bacteria
that do not harbor the kanamycin resistance gene cannot synthesize protein in the presence of
kanamycin and die. The kanamycin resistance gene encodes an enzyme that uses kanamycin as its
substrate, modifies it, and inactivates it. Thus if the bacteria that has been transformed with the
expression plasmid is grown in culture medium that contains kanamycin, cells that accidentally lose the
expression plasmid will be killed. So growth of the transformed bacteria in the presence of kanamycin
ensures that the expression plasmid is maintained in all cells in the culture.
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Figure 21 Expression of LuxG in Tuner(DE3) E. coli. Tuner(DE3) E. coli has a modification where a version of the lac operon has
been incorporated into the bacterial genome by  phage infection. The version of the lac operon that is incorporated has the
lac promoter and operator sequences, but the downstream genes have been replaced by T7 RNA polymerase. Regulation of
gene expression for LuxG occurs as follows. Left: LacI, the lac repressor, is normally expressed by E. coli. LacI binds to the
operator and prevents E. coli RNA polymerase from binding to the promoter. With no T7 RNA polymerase, transcription from
the T7 promoter does not occur and LuxG expression remains off. Right: In the presence of lactose, lactose binds to LacI and
prevents it from binding to the operator. E. coli RNA polymerase recognizes the promoter and T7 RNA polyermase is
expressed. T7 RNA polymerase recognizes its promoter on the pGhis expression plasmid and transcribes the LuxG gene. Note
that for this exercise, you will be using the lactose analog, IPTG, instead of lactose.
The remaining components of the expression plasmid are related to expressing and purifying the protein
of interest. Expression of the luxG gene is under the control of the T7 promoter which initiates
transcription in the presence of T7 RNA polymerase. T7 is a bacteriophage (virus that infects bacteria)
and like most viruses, their promoters tend to direct high levels of expression (i.e. they are strong
promoters). Usually the viral promoter directs the expression of viral proteins but the expression vector
we will use has luxG downstream of the T7 promoter which has been placed there through recombinant
DNA techniques. This arrangement ensures a high level of expression of luxG since the T7 promoter is a
strong promoter.
T7 RNA polymerase is incorporated into the E. coli genome by using another bacteriophage,  phage. 
phage is capable of undergoing a life cycle stage known as lysogeny, meaning the viral DNA can insert
itself into the bacterial chromosome. Taking advantage of this lysogenic DNA insertion capability, a DNA
construct that encodes the T7 RNA polymerase under the control of the lac promoter is inserted into
the E. coli chromosome. This lac promoter controlled T7 RNA polymerase construct was named “DE3.”
Thus the E. coli that you will be using carries the designation “DE3”.
The lac promoter is something you probably already have learned about. Many general biology courses
cover gene regulation and use the lac operon as an example. This operon consists of a promoter and
operator sequences that are controlled by the presence of lactose. In the absence of lactose, a protein
called the lac repressor (LacI) binds to the operator. LacI inhibits transcription of downstream genes by
preventing E. coli RNA polymerase from binding the promoter to initiate transcription. In the presence
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of lactose, lactose binds to the LacI,
inhibiting it from binding to the operator
site. Under this condition, transcription is
able to occur and downstream genes are
expressed. Normally, the downstream
genes encode those useful for lactose
metabolism. However, the genetic
construct has been modified in E. coli
carrying the DE3 designation. The genes
that are normally used for lactose
metabolism have been replaced by the T7
RNA polymerase gene. The resulting
system is presented in Figure 21.
Lactose is a sugar that can be used to
initiate expression of LuxG in the system
you will be using. However, lactose is also
naturally metabolized by bacteria.
Therefore to use lactose for inducing
expression of LuxG, lactose would have to
be added at regular intervals to
compensate for the rate at which it is
Figure 22 Bacterial growth curve.
metabolized. For this reason, researchers
use the lactose analog, isopropyl -D-1thiogalactopyranoside (IPTG). This analog can bind to and inhibit LacI and is not metabolized by
bacteria, allowing continuous gene expression following a single addition of IPTG.
The experiment, induction of LuxG expression
Expression of heterologous genes in E. coli follows the same general protocol regardless of what gene is
expressed. E. coli cells that harbor the expression plasmid are generated by transformation and stored
in small volumes (~0.5 mL) in the -80oC freezer in the presence of some cryoprotectant such as glycerol.
When a protein expression experiment is to be undertaken, culture media is sterilized in an autoclave in
Erlenmeyer flasks. Standard culture media is Luria Broth (LB) which consists mostly of digested proteins,
yeast extract, and salt. The cultures are cooled and selection antibiotics (like kanamycin) are added.
Using sterile technique, bacteria are grown overnight at 37oC on a petri dish that has sterile LB/KAN that
has been solidified using agar. E. coli is an intestinal bacterium so its optimal growth temperature is
37oC. Following overnight incubation, individual bacteria multiply into visible colonies on the plate.
Several of these colonies are used to inoculate the sterile liquid LB/KAN cultures.
All of these steps will be completed when you enter lab. Several cultures will have been inoculated in
the morning and will be growing at 37oC under vigorous shaking. Shaking ensures a constant supply of
oxygen which encourages growth since E. coli is a facultative anaerobe, meaning that it can grow in the
absence of oxygen, but grows better when able to conduct oxidative phosphorylation in the presence of
oxygen.
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You will be responsible for inducing the expression of LuxG by adding IPTG. IPTG cannot be added at
anytime, however. As bacteria grow in a culture, it enters several growth phases. The first phase is the
lag phase. This is the initial growth phase where the bacterial concentration is very low. There are
plenty of nutrients so the bacteria are actively synthesizing protein and grow quickly. Addition of IPTG
at this early stage would result in low protein yields since the cell density is low.
In the next stage of growth, the log phase, bacterial cell density is high and the nutrient levels are still
high enough to support rapid growth. This is the ideal point to add IPTG since the bacteria in the culture
are actively synthesizing protein during rapid growth and there are plenty of bacterial cells that will be
expressing protein.
The last stage of the culture is the stationary phase. At this stage, the bacteria are at a very high cell
density, but the nutrients have been mostly used. Since the bacteria no longer have nutrients, the
amount of protein synthesized is reduced. Induction of protein expression at this stage yields low
protein expression because the cells lack the nutrients to support the expression.
The ideal stage to add IPTG is during log phase growth as summarized in Figure 22. You will monitor
when your culture has reached log phase by periodically taking readings of the OD600, or optical density
at 600 nm. As the culture grows and moves through the growth phases, it becomes increasingly turbid,
or cloudy. This phenomenon occurs because bacterial particles that are in suspension scatter light. If
placed in a spectrophotometer, the turbidity of the culture scatters the incident light and causes an
apparent absorbance reading. To distinguish between absorbance of light by molecules dissolved in
solution and light scattering caused by particles in suspension, the former is referred to absorbance and
the latter is called optical density. Both are measured the same way using a spectrophotometer.
Empirically, it has been determined that log phase growth is reached for a culture of E. coli when the
OD600 of the culture equals 0.6. As described above, it is important not to miss log phase growth since
doing so will likely reduce your yield. To estimate when your culture will reach log phase, it is useful to
keep in mind that the doubling time for E. coli is about 30 min. Therefore, if your culture has an OD600
reading of 0.3, you should plan on checking the culture again in 30 min since it will have doubled (in
theory) and the OD600 should then read 0.6.
Why bother with induction with IPTG?
Catching the right time to induce the culture may seem like a timing hassle. Why bother with an
inducible system, anyway? The ultimate goal is the produce the protein of interest so one could go
about expressing the protein using a constitutive promoter, or one that is always on. There are a couple
of reasons why constitutive promoters are not typically used in bacterial expression systems. One
reason is that the protein being expressed may be somewhat toxic to the cells. Using an inducible
system in this case enables the culture to be grown. Once grown, sufficient amounts of protein can then
be induced to express before the cells die from the induced protein’s toxicity. However even in the case
where the protein of interest is not significantly toxic to the E. coli, an inducible system is still desired.
Keep in mind that the T7 promoter is a strong viral promoter, meaning that it will transcribe the gene at
a high rate. This quality is strongly desired for high levels of protein expression, but it also means that
the cells become unhealthy because much of their protein synthesis activities become directed toward
expressing the protein of interest and not the normal proteins that are needed for cell survival. In light
of this, the T7 promoter is a double-edged sword. It encourages expression of a large amount of the
protein of interest, but it makes the culture unhealthy. This is why an inducible system is needed. If a
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strong promoter is desired for high level expression, that promoter needs to be kept inactive until the
culture reaches log phase. Otherwise the culture will always be unhealthy and will be difficult to grow
to high densities.
Inclusion bodies and protein expression temperature
After adding IPTG, the protein that is under the control of the T7 promoter will be expressed. In your
experimental system, LuxG will be expressed. Obviously, this is an unnatural system and for some
overexpressed proteins this seems to be recognized by E. coli. E. coli sometimes places overexpressed
proteins into inclusion bodies. Inclusion bodies are insoluble and misfolded proteins that are not active.
Researchers try to avoid inclusion body formation since recovering activity requires refolding the
protein, a process that requires time consuming screening of buffers, salt, reducing agents,
temperature, etc. to find the correct refolding conditions.
Though not thoroughly understood why, lowering the temperature after inducing T7 promoter driven
protein expression with IPTG often reduces the amount of inclusion body formation. One likely
explanation is that the rate of protein translation might play a contributing factor. At lower
temperatures, reaction rates are slowed, thus perhaps slower rates of protein synthesis limit inclusion
body formation.
Cell Lysis
E. coli is a bacterium. Unlike mammalian cells, such as those from the bovine tissue we had worked with
for LDH purification, bacteria have a peptidoglycan cell wall. This cell wall makes bacteria more difficult
to lyse to obtain the cell extract. A common method for lysing bacteria is to weaken the peptidoglycan
cell wall with the enzyme, lysozyme. Following lysozyme treatment, the cell membranes are disrupted
by sonication. During sonication, high frequency sound waves are imparted into the harvested bacterial
cells which causes small areas of high and low pressure within the cell suspension. These changes in
pressure provide enough force to break open the bacterial cell membranes.
Nijvipakul, S et al. (2008) J Bacteriology 190: 1531–1538
References
Hosseinkhani, S, Szittner, R and Meighen, EA (2005) Random mutagenesis of bacterial luciferase: critical
role of Glu175 in the control of luminescence decay. Biochem. J. 385: 575–580
Ingelman, M, Ramaswamy, S, Nivie`re, V, Fontecave, M and Eklund, H (1999) Crystal Structure of
NAD(P)H:Flavin Oxidoreductase from Escherichia coli. Biochemistry 38: 7040-7049.
Nijvipakul, S, Wongratana, J, Suadee, C, Entsch, B, Ballou, DP and Chaiyen, P (2008) LuxG Is a Functioning
Flavin Reductase for Bacterial Luminescence. J Bacteriology 190: 1531–1538
Song, S. H., Dick, B., Penzkofer, A., and Hegemann, P. (2007) Photo-reduction of flavin mononucleotide
to semiquinone form in LOV domain mutants of blue-light receptor phot from Chlamydomonas
reinhardtii, J Photochem Photobiol B 87: 37-48.
LuxG Introduction
Techniques in Protein Chemistry
CHEM 108
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Winfrey, MR, Rott, MA and Wortman, AT (1997) Unraveling DNA: Molecular Biology for the Laboratory.
Pearson/Prentice Hall.
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Techniques in Protein Chemistry
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Exercise 1 Expression of LuxG-his6 in E. coli and Release by Lysis
Reagents, Supplies & Equipment
luxG construct (pGhis): Protein coding sequence (CDS) of luxG from Photobacterium leiognathi with a
hexahistidine tag at carboxy end. Vector = pET24b (5,309 bp. Includes lacI .)
Insert = 717 bp. Entire plasmid = 5,942 bp. Resistance = kanamycin.
Host from EMD Bioscience/Novagen: Tuner(DE3)
F–ompT hsdSB(rB– mB–) gal dcm lacY1 (DE3)
Sterile LB broth with kanamycin antibiotic (30 g/mL): 200 mL in a 1 L Erlenmeyer flask. IPTG (induces
expression): MW = 238 g/mole at 100 mM (24 mg/mL) in water, filter sterilized. PMSF (proteinase
inhibitor): 10 mg/mL (60 mM) stock in EtOH. MW = 174 g/mol. Stored at -200. Nondenaturing
Resuspension Solution: 50 mM Tris-8
100  (17 g/mL) PMSF, 10 % glycerol, rLysozyme (recombinant, from Novagen): 30,000 units/L,
Bradford staining reagent: 0.01 % (w/v) Coomassie Brilliant Blue G-250 4.75 % (v/v) ethanol, 8.5% (v/v)
phosphoric acid, Protein standard for Bradford assay: bovine -globulin (IgG), 0.1 or 1 mg/mL.
Screw-cap tubes and bottles, Centrifuge bottle (250 mL) with rubber O-ring in cap (to ensure tight seal).
Low-speed ( < 6,000 rpm) spins to pellet cells. "15 mL Conicals": Clear plastic, thin (1 mm) wall with
conical bottom (disposable). Low-speed (2,000 rpm) spins and sonication, Oak Ridge tubes: Vol = 38 mL.
Opaque plastic, thick (2-3 mm) wall with round bottom. High-speed (13,000 rpm) spins (reusable, there
should be two in your locker).
Spectrophotometry supplies
Bacterial cultures: Disposable glass culture tubes (13 x 100 mm) or plastic cuvettes (1 mL). Bradford
assay: Glass culture tubes (Spec 20) or multiwell plate (Appendix V). Branson 450 Sonifier
Spectrophotometers: Spectronic 20D+ (Spec 20), FLUOstar Optima plate reader, Amersham or Perkin
Elmer UV/vis spectrophotometer. Markers for labeling tubes to be quick-frozen in dry ice/isopropanol:
VWR or Fisher brand, alcohol/waterproof, black ink (blue or other colors will wash off).
Precautions
Using the JA-14 rotor in the preparative centrifuge: Your TA should closely supervise to be certain the
sample bottles are balanced and the rotor is secure before the spin is initiated.
Using sonifier: The high frequency can damage your ears. Wear the ear protection.
Procedure
Day 1: Express LuxG-his6, Pellet and freeze cells next day
Growth and induction with IPTG
The Tuner/pGhis cell cycle is 30 min at 370C but 7 h at 160C A culture of 1 x 108 cells/mL has an OD600 =
0.6.
LuxG Exercise 1 Expression of LuxG-his6 in E. coli and Release by Lysis
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The culture will be grown at 370C until the OD600 = 0.5-0.6 (induction density), at which time the inducer
(IPTG) will be added. The incubation will then be shifted to 16oC and allowed to continue until the next
morning. The OD600 should increase during this incubation.
1) At ~8:00 AM a 250 mL LB/kanamycin culture will be inoculated with three colonies (~1 mm) of
Tuner/pGhis and incubated at 370C with shaking.
a. This step will be taken care of for you
2) During your lab period, begin assaying absorbance (~4 hours post-innoculation).
a. It will probably be most convenient to use the Amersham or Perkin Elmer specs and
disposable 1 mL cuvettes. Since you will not be measuring at an ultraviolet wavelength,
you can use a cuvette made of normal plastic. Alternatively, the Spec 20 and glass tubes
may be used.
3) When the induction density is attained, save the last cuvette of bacteria and cool the flask on an
ice+water bath.
a. The temperature should be at or below 16oC.
b. While the flask is cooling, spin down 1 mL of the bacteria aliquot in an eppendorf tube,
labeled “LuxG Preinduction,” and your name. Remove the supernatant and store the
bacterial pellet in the -20°C. This sample does not require snap freezing.
4) Add IPTG to the chilled culture to 0.4 mM (aseptic technique!).
5) Grow at 16o C until the next morning to allow expression.
The next day (outside of normal lab period):
6) Assay OD600 again to confirm growth during induction.
7) Obtain a centrifuge bottle to spin the culture
a. Measure and record the empty bottle’s mass.
8) Transfer the culture to the centrifuge bottle and balance with another similar sample. Be certain
the cap of the centrifuge bottle is tight enough to prevent leakage during centrifugation. You
definitely want to avoid contaminating the rotor with bacteria!
9) Spin down cells: 6,000 rpm in JA-14 rotor (5,500 x g) for 20 min at 4oC.
10) Dispose the supernatant into the appropriate waste container
11) Measure the mass of the centrifuge tube plus the bacterial pellet.
12) Resuspend the pellet (on ice) in 10 mL of: 50 mM Tris-8, 100 M (17 g/mL) PMSF, 10 % glycerol
(glycerol helps to keep LuxG soluble).
13) Transfer to a 15 mL, screw-cap, conical bottom tube.
a. Do not use a larger tube! The 15 mL size is the best for sonication. Label tube with ink
that will not wash off in alcohol. Do not use tape, it falls off in the freezer.
14) Freeze in dry ice/isopropanol and store at -200C until ready for sonication
a. -20oC freezer is sufficient for storage of all LuxG-his6 or LDH samples in this course. In
most laboratories, protein samples are stored at 80oC. Many -20oC freezers go through
a defrost cycle where the contents warm. Also, contents are more likely to thaw when
the door is open in a -20oC freezer.
Day 2: Lyse Tuner/pGhis Cells and Assay Protein Concentration
Enzymatic digestion of cell walls.
A combination of enzymatic and mechanical disruption will be used to lyse the E. coli cells. After the
cells are thawed, the lysozyme enzyme is added to digest the proteoglycan cell wall. Further breakdown
LuxG Exercise 1 Expression of LuxG-his6 in E. coli and Release by Lysis
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of the cell wall and disruption of the plasma membrane is then achieved by exposing the preparation to
pulses of high frequency sound with a sonifier. A centrifuge spin is performed to pellet the fragments of
cell wall and membrane (and proteins linked to these structures). The soluble LuxG-his6 enzyme remains
in the supernatant.
1) Calculate the mass of the bacterial pellet
2) Novagen recommends 5,000 units of rLysozyme per gram of cells. Calculate the volume of
rLysozyme you will need to add to your culture.
3) Remove the resuspended bacteria from the freezer and allow to thaw in a beaker of cool water.
You may change the water in the beaker to accelerate the thawing process, but do not use hot
water.
4) Add the appropriate amount of rlysozyme and incubate at 30 oC for 15 min.
Sonication (wear ear protection)
This step is a bit tricky. If it doesn't work well it will be difficult to complete the remaining LuxG
exercises. It is critical that: 1) At least 90% of the bacteria are lysed, 2) splashing should be minimized so
that the cells are consistently exposed to the full power of the sonifier and to avoid loss of sample and 3)
the enzyme is not denatured by increase in temperature caused by sonication.
The sonifier should be set to do a set of 20 pulses as follows:
Amplitude = 50% (50 Watts).
Duty cycle: pulse on = 0.7 s, pulse off = 0.3 s.
1) Following lysozyme treatment, place the sample in a beaker of ice with a little bit of water (it
should not be slushy).
2) Place the end of the sonifier horn below the level of the liquid of your resuspended bacteria.
a. While sonicating, do not allow the horn to touch the sides of the tube or allow the end
of the horn to become exposed outside of the culture.
b. Be prepared to immediately halt the sonifier if the resuspended bacteria begins to froth.
3) Run the sonifier program, then leave on ice for at least 10 min to bring temperature back down
4) Repeat the sonication steps to help ensure complete lysis.
5) Save a 100 L aliquot of the “Whole Cell Lysate” in the -20oC freezer.
6) Transfer the sample to a 38 mL Oak Ridge tube, on ice.
7) Centrifuge the sonicated lysate to remove membranes and membrane-associated proteins:
a. Spin at 13,000 rpm in JA-14 (26,000 x g), 20 min, 4oC. Measure the volume of the
supernatant and transfer to a clear-plastic, 15 mL conical tube. The solution should be
pale yellow. If it shows no color, the cells were not lysed and the pellet should be
resuspended in lysis buffer and the sonication process should be repeated.
8) The clarified lysate is the “Crude Extract”.
a. Measure and record the volume of the crude extract.
Examine the pellet for inclusion bodies. Foreign proteins expressed in E. coli sometimes become
sequestered in these structures if the tertiary structure has not formed properly. The membrane/cell
wall portion of the pellet will be light brown whereas inclusion bodies are white. The pellet will be
discarded because any LuxG-his6 in it is unlikely to have activity, but you will save an aliquot to analyze
by SDS PAGE. Even if some of the enzyme is in the pellet, the majority should be in the supernatant. Still,
it is often useful to examine the inclusion body content by SDS-PAGE.
LuxG Exercise 1 Expression of LuxG-his6 in E. coli and Release by Lysis
Techniques in Protein Chemistry
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Prepare the inclusion body sample for SDS-PAGE:
1) While being careful not to disturb the pellet, wash the pellet with water.
2) Discard the water
3) Measure a volume of water that equals that recorded for the Crude Extract
4) Add the water to the inclusion body pellet
5) Create an inclusion body suspension by vigorous mixing. You may need to use mechanical force
(e.g. use a spatula).
6) The preparation will not be completely even (it will be a suspension, not a solution), but when
you’ve done the best you can, pipet 120 L into an eppendorf tube and store at -20oC.
Prepare the crude extract samples:
1) Save an 80 L aliquot of the crude extract for Bradford analysis
2) Save a 120 L aliquot for SDS-PAGE analysis.
3) Save two 0.5 mL aliquots for activity assays.
a. For preserving activity, those aliquots saved for activity assays should be snap frozen in
an isopropanol/dry ice bath
b. Glycerol does not need to be added at this point (Can you determine why?)
4) Store the samples in the -20oC freezer
5) The remaining extract will be used for the Ni affinity chromatography. Snap freeze the
remainder and store in the -20oC freezer.
References
Bio-Rad (2003) Protein quantitation: Quick start Bradford protein assay. Tech bulletin 2969.
http://www.bio-rad.com
Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-54
Nijvipakul, S et al. (2008) LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence
J Bacteriology, 190: 1531–1538
pET-24a-d(+) Vectors (1998) Novagen Tech bulletin 070.
pET System Manual (2006) 11th ed., Novagen Tech Bulletin 055 Expressing the Target Gene, pp 19-24
Target Protein Verification: Soluble Cytoplasmic Fraction, 28-30 Additional Guidelines: Choosing a pET
Vector, 37-45 Regulating Protein Expression in the pET System, 45-48
Hosts for Expression, 49-54
Studier, FW et al. (1990) Use of T7 RNA Polymerase to Direct Expression of Cloned Genes Meth Enz, 185:
60-89.
Studier, F.W. (2005) Protein production by auto-induction in high-density shaking cultures
Protein Expression and Purification 41: 207–234
Regulation of the lac operon in E. Coli
Lewin, B. (2008) Genes IX, Chapter 12 (pub: Jones & Bartlett)
Watson, J.D. et al. (2008) Molecular Biology of the Gene, 6th ed., Chap 16, pp 553-566. (pub: Pearson
Ed.)
LuxG Exercise 1 Expression of LuxG-his6 in E. coli and Release by Lysis
Techniques in Protein Chemistry
CHEM 108
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Exercise 2 Affinity Purification of LuxG-his6 with Nickel Column
When designing and constructing an expression vector, fusion tags are frequently added as a
modification to the protein of interest’s sequence. These fusion tags are proteins or small peptides that
are not native to the protein of interest’s natural sequence, but play an important experimental role.
They are almost exclusively placed at the N- or C- terminus of the protein to limit any effect they might
have on the protein’s normal function and are incorporated as part of the DNA sequence when the
expression vector is constructed. Some functions of fusion tags include serving as an epitope tag for
detection of the protein by Western Blot or some other immunological tagging method. Another
common function is to serve as an affinity tag that can be recognized by a resin.
A widely used affinity tag is a poly-histidine tag which usually consists of a series of at least six histidine
amino acids. Part of the LuxG expression vector encodes a poly histidine tag where six histidines are
placed at the C-terminus. The imidazole ring on the histidine side chains have an affinity for Ni 2+ ions.
When Ni2+ is immobilized on resin, it can provide a means for affinity column chormotography.
The crude extract from the LuxG expression will be applied to the Ni resin. Most of the host cell (E. coli)
proteins should not bind the column and should flow through the column. Some host E. coli proteins
will have weak affinity for the resin if they have a high frequency of histidine in their sequence. Washing
with a low concentration of imidazole will wash these proteins off of the resin. After washing the
column, his-tagged LuxG is eluted with a buffer that is high in imidazole concentration.
The Ni-column is sensitive to certain chemicals that are commonly used during protein purification so
care should be used when designing the experiment. Recall for LDH, we used EDTA in the buffer as a
protease inhibitor. EDTA is a strong metal chelator. If added to the crude extract for LuxG, the EDTA
will bind to the Ni2+ on the resin and cause it to elute. Additionally, reducing agents such as ME and
DTT usually serve to stabilize LuxG since LuxG is normally expressed the cell cytosol which is a reducing
environment. However reducing agents tend to reduce the Ni 2+ on the column, weakening the
interaction with the resin and causing the Ni+ to then elute from the column. For these reasons, EDTA is
not added as a protease inhibitor and reducing agents are not added until after the protein has eluted
from the column.
Reagents, Supplies & Equipment
Spectrophotometers: Spectronic 20D+ (Spec 20) or FLUOstar Optima plate reader.
Plastic column: inside dia = 1 cm, height = 10 cm (no tubing or fraction collector necessary). Ultra-4 10K
Centrifugation/Filtration Device [from Amicon, 10K is the nominal molecular weight limit (NMWL),
refer to appendix VI].
Bradford assay supplies: Culture tubes or multiwell plate.
His-bind resin from EMD Biosciences/Novagen: Uncharged iminodiacetic acid (IDA) agarose resin. The
binding capacity is approximately 8 mg/ml bed volume.
50 mM NiSO4
Binding, wash, and elution buffers:
50 mM Tris pH 8
250 mM NaCl
LuxG Exercise 3 SDS-PAGE of Tuner/pGhis Lysate and Purified LuxG-his6
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10% glycerol
and imidazole at: 20 mM (binding), 50 mM (wash) or 250 mM (elution)
1 M dithiothreitol (DTT)
Bradford assay reagents: protein standard and staining reagent as in LDH Exercise 1.
Procedure
Expect ~4% of Tuner/pGhis lysate protein mass to be LuxG-his6. A packed resin volume (“bed volume”)
of 1 mL should be sufficient.
Set up the column in the cold room.
The following may have been done for you already. If the resin is greenish-blue, skip this section
Mark the 1 mL level on the column with a Sharpie by filling the column with 1 mL of water
Remove the water
Fill the column with resin such that the bed volume is at approximately the 1 mL level.
Note that the Ni resin slurry is a mixture of resin and buffer. This means you will need to add >1
mL of the slurry to leave a bed volume of 1 mL.
You may need to remove some of the resin if you have too much; check with your TA.
*** Never discard the resin *** All resin should be saved for reuse.
Allow the liquid to flow out from the bottom of the column. The level of the remaining resin should be
at the 1 mL mark.
Add 5 mL of water to wash out the storage buffer in the resin
Charge resin with the NiSO4 solution
Add 2 bed volumes (2 mL) of the NiSO4 solution
Wash with water
Add 5 bed volumes of water
Equilibrate with binding solution.
Add 10 bed volumes of binding solution.
The column is now ready to receive the cell lysate containing the LuxG his-tagged protein.
1) Apply the initial supernatant to the column and collect the flow through
a. Fractionate into 2 tubes
2) Save an aliquot of the second flow through fraction for running on an SDS PAGE gel. The sample
can be stored in the -20°C freezer next to the initial supernatant. The sample does not need to
be snap frozen.
3) Add 6 bed volumes of binding solution to push most of the non-tagged protein through.
4) Wash with 5 bed volumes of wash solution to ensure that all nontagged protein has been
removed.
5) Elute tagged protein with 5 bed volumes of elution solution, collecting 1 mL fractions.
a. Expect tagged protein to be distributed in approximately 3 fractions.
b. Add DTT to the elution fractions to 1 mM.
6) Close stopcock so that the resin does not dehydrate (lab manager will remove resin for reuse).
7) Determine which elution fractions contain protein
a. Start by taking A280 measurements
LuxG Exercise 3 SDS-PAGE of Tuner/pGhis Lysate and Purified LuxG-his6
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
8)
9)
10)
11)
12)
13)
14)
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b. If A280 is not sensitive enough, conduct a quick Bradford.
i. Conduct the Bradford assay as described in LDH Exercise 7 using 60 L from
each fraction.
ii. Do not make a standard curve, you will make a more accurate measurement
later
Increase the concentration of affinity-purified protein by centrifugation/filtration:
a. The Ultra-4 will accept up to 4 mL of solution. If the volume of affinity-purified protein is
greater than this, do multiple loadings. A detailed description of the use of these devices
is in the Appendix. The concentrator consists of an upper and lower chamber, separated
by a porous membrane. The membrane pores are very small, so small in fact that they
do not allow large molecules to pass through. For the 10K NMWL concentrator
molecules larger than 10,000 Da (on average) cannot pass through. The sample you wish
to concentrate is placed in the upper chamber and the concentrator unit is centrifuged.
Because LuxG’s MW is greater than the NMWL of the device, all of the LuxG-his6 should
remain in the upper chamber, while water, buffer, salts etc. are small and pass through
the filter. Since the amount of protein in the upper chamber remains the same while the
volume in the upper chamber decreases after the centrifuge spin, the concentration of
the protein is increased. The goal is to reduce the volume to ~10% of the original value
(3 mL will be concentrated to 0.3 mL).
Add the protein sample to the upper chamber and spin at 7,000 rpm (7,500 x g in the JA-14
rotor) for 10 min at 4oC.
If the volume has been decreased sufficiently, use a P-200 to mix, then transfer it to a test tube.
If the volume still needs to be reduced, mix the protein in the concentrator and do another spin
for an appropriate length of time before recovering the protein sample.
a. Why should you mix? Imagine what is physically occurring near the membrane. Protein
is getting pushed up against the membrane, but is not able to pass through. Therefore
there will be a large amount of protein next to the membrane after the spin. To recover
this protein rich portion, use the pipette to mix up the area by pipetting up and down
before recovering the protein sample.
Record the final volume.
Assay the protein concentration in the affinity column fraction. Also assay the concentration of
the crude extract aliquot that was stored in the freezer from LuxG Exercise 1.
a. Use a Bradford assay with standard curve as described in LDH Exercise 7.
b. As a starting point for the crude extract, try 30 and 60 L of a 1:10 dilution.
c. If using a multiwell plate, assay 10 L of the column fraction in 200 L reactions. If using
the Spec 20, start by assaying 6 L of the column elution fraction in 3 mL reactions (you
may have a better estimate based upon the quick Bradford attempted above. Don’t use
an A280 for an estimate if you have < 1mL).
Calculate the concentration of pooled protein.
Split the concentrated protein into four equal aliquots, add glycerol to ~10%, and freeze in dry
ice/isopropanol and store at -20oC.
Data Analysis
Analyze the Bradford results
Prepare a table of the protein concentrations and total protein content for your samples.
References
LuxG Exercise 3 SDS-PAGE of Tuner/pGhis Lysate and Purified LuxG-his6
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 99
Fall 2018
Boyer (2006) Production of proteins by expression of foreign genes. In: Biochemistry Laboratory,
Modern Theory & Techniques. Pearson/Benjamin Cummings. pp 339-344.
His-bind Kits (2006) Novagen Tech Bulletin 054. pp 2-5, 8-10.
Exercise 3 SDS-PAGE of Tuner/pGhis Lysate and Purified LuxG-his6
This type of electrophoresis is also described in: SDS-PAGE of the Initial Supernatant and Purified LDH.
SDS = sodium dodecyl sulfate (a detergent) and PAGE = polyacrylamide gel electrophoresis. Proteins are
denatured by boiling in SDS. The hydrophobic dodecyl structure binds at various sites along the
polypeptide chain while the sulfates provide an excess of negative charge. SDS is also included in gel and
the running buffer assuring that the proteins remain coated with detergent during electrophoresis. Since
SDS-coated proteins are “anionic” they migrate to the anode (positive terminal) of the electrophoresis
apparatus.
Incorporation of SDS into protein causes denaturation, disrupting quaternary structure, i.e. the
noncovalent association of subunits. Thus, enzymes that function as multimers in cells migrate as
monomers on these gels. Since denaturation also eliminates secondary and tertiary structure, the rate
of migration is solely dependent on molecular weight (MW). Larger proteins migrate more slowly than
smaller ones because they have more contact with the polyacrylamide matrix. A semilog plot of
log10(protein MW) as a function of distance migrated gives a straight line for proteins whose MW is
within the resolving range of a particular concentration of polyacrylamide.
Reagents, Supplies & Equipment
Electrophoresis power supply.
Gel casting supplies as described in appendix IV. Electrophoresis apparatus with electrode connectors.
10% β-mercaptoethanol, 50 mM sod. phos., pH 7.2 80% glycerol, 0.25% bromophenol blue
10% SDS (100 μg/μL)
10x Tris-glycine/SDS running buffer
Reagents for SDS polyacrylamide gel preparation (shown below, in appendix I and in the SDS-PAGE
exercise in the LDH section).
Gel stain: Simply Blue (Invitrogen): Coomassie Brilliant Blue G-250 in a nonorganic solvent. 20% NaCl: to
be added to the Simply Blue.
Electrophoresis Markers: Standard Polypeptides, 0.5 g/L, run 5 g. Names of the proteins are in Data
Analysis below. Note to TAs: stock will probably be 5 g/L so dilute it before distributing.
Precautions
The electrophoresis apparatus has a safety interlock but you still need to be careful about avoiding any
direct contact with electrical current.
LuxG Exercise 3 SDS-PAGE of Tuner/pGhis Lysate and Purified LuxG-his6
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Stain solution should be handled with gloves. Although it does not contain an organic solvent, it is still
best to place overnight staining/destaining trays the in fume hood.
Procedure
The separating and stacking gel recipes for LuxG electrophoresis are below. Assemble the plates and
spacers in the casting stand (as described in appendix IV) before mixing the gel components. The
solution must be poured between the plates immediately after adding the catalysts (APS and TEMED).
Expected Migration of Tagged LuxG
LuxG has 237 amino acids and the molecular weight (MW) = 26,300. The MW of his6 = 930. Therefore
MW of LuxG-his6 = 27,230.
1) Mix the following ingredients for the 15 % separating (sep) gel:
5 mL of 30% acrylamide, 0.8% bisacrylamide.
2.5 mL 4x Tris hydrochloride buffer for SDS separating gel.
2.5 mL H2O.
100 L of 10% APS
10 L TEMED
2) Prepare Samples for Electrophoresis
Tube
Protein Sample
Amount
(g)
10% mercaptoethanol
10 mM sodium
phosphate, pH 7.2
(L)
80% glycerol 10%
0.25%
SDS
bromophenol (L)
blue (L)
1
2
(N/A)
Standard
Preinduction
10
20
1
3
4
5
6
(N/A)
120
240
(N/A)
Whole Cell Lysate
Crude Extract
Crude Extract
Inclusion Body
10
10
10
10
20
20
20
20
1
1
1
1
7
(N/A)
Flow Through
10
20
1
8
(N/A)
Flow Through
10
20
1
9
10
4
20
Affinity Purified
Affinity Purified
10
10
20
20
1
1
Protein
(L)
Distilled
H20
(bring
volume
to 50 L)
(L)
5
See
below
50
Same
vol as
Tube 4
Same
vol as
Tube 4
Same
vol as
Tube 5
For the Preinduction sample, add the components as described to the sample tube. The tube should
only contain a cell pellet so the final volume will be ~100 L.
3) Just before loading on gel:
a. Heat samples in boiling water bath for 60 s, then spin to bring all liquid to bottom of
tube.
LuxG Exercise 3 SDS-PAGE of Tuner/pGhis Lysate and Purified LuxG-his6
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b. Check the preinduction tube. If it is particularly viscous, vortex vigorously and centrifuge
at max speed for 10 min.
4) Mix the ingredients for the 4.5 % stacking gel:
1.25 mL 4x Tris hydrochloride buffer for SDS stacking gel.
0.75 mL 30% acrylamide, 0.8% bisacrylamide.
3.0 mL water.
35 L 10% APS
8 L TEMED
Electrophoresis
1) Dilute 10x Tris-glycine running buffer to 1x and set-up the gel system
2) Load the samples
3) Run at 200 V. Run until dye is at bottom (~45 min), run longer for it to reach 1cm from the
bottom plate.
Staining, Destaining, and Recording the gel image
1) Stain the gel with Simply Blue plus NaCl using the microwave method as done previously.
2) Capture the gel image using the digital cameras as done previously.
Data Analysis
1) A semilog plot of standard protein migration must be created.
a. Calculate the log10(MW) for each standard. Using Excel, plot these values on the y-axis
and the distance migrated on the x axis.
2) Use the standard curve to calculate the molecular weight of LuxG-his6 monomer by
interpolation on the semilog plot.
References
Boyer, R (2006) Discontinuous gel electrophoresis. In: Biochemistry Laboratory, Modern Theory &
Techniques. Pearson/Benjamin Cummings. pp 181-184.
Davis, B.J., (1964) Disc electrophoresis II. Method & application to human serum proteins. Annals NY
Academy Sci 121: 404-427.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage
T4. Nature227: 680-685.
Ornstein, L (1964) Disc electrophoresis I. Background & theory. Annals NY Acad Sci 121: 321-49.
Tulchin, N, Ornstein, L and Davis, BJ (1976) A microgel system for disc electrophoresis.
Anal Biochem72:485-490.
LuxG Exercise 3 SDS-PAGE of Tuner/pGhis Lysate and Purified LuxG-his6
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 102
Fall 2018
Exercise 4 Flavin Reductase Activity Assays
Reagents, Supplies & Equipment
Spec 20 spectrophotometer.
Disposable glass culture tubes (13 x 100 mm) for assaying reactions in Spec 20.
Reaction Buffer
50 mM Tris-8, 10% glycerol, 1 mM DTT (may also be used later for desalting column)
Substrates
disodium-NADH: MW = 709.5, 340 = 6.22 mM-1cm-1, stock is 20 mM.
sodium-FMN: MW = 478.4, 450 = 12.2 mM-1cm-1, stock is 4 mM
riboflavin: MW = 376.37, 450 = 11.3 mM-1cm-1, stock is 400 M
The  for the flavins is provided as a reference. Only the  for NADH is used to calculate the reductase
activity.
Procedure
Do this assay on aliquots of: the Crude extract and the affinity purified LuxG-his6. Reaction volume
should be ~3 mL (minimum for reading in Spec 20).
1) For both of the samples to be assayed, calculate the [LuxG-his6] in M (use 27,230 as the MW).
For the initial supernatant, take the mass of protein obtained from the affinity purification and
divide by the volume of the crude extract. This should be the approximate concentration of
enzyme in the initial supernatant. Convert mg/mL to M.
2) Calculate the volume of each of the three samples to add to a 3 mL reaction to give a final
enzyme concentration of 0.4 M.
3) The assay mixes are shown in the table.
4) Add the enzyme to initiate the assay then begin taking A 340 timepoints.
a. Record the A340 of the reaction a zero time point and then every 30 s for at least 3 min
(Just like the LDH assay).
Component
Condition 1
Condition 2
Condition 3
Condition 4
Lysate or pure
protein
NADH
FMN
Riboflavin
Reaction
buffer
Total Volume
(variable)*
(variable)*
(variable)*
--
0.03 mL
0.03 mL
-2.94 mL
0.03 mL
-0.30 mL
2.67 mL
0.03 mL
--2.97 mL
0.03 mL
0.03 mL
0.30 mL
2.64 mL
3 mL
3 mL
3 mL
3 mL
Final
concentration
0.4 M LuxGhis6
200 M
40 M
 M
A340: Should be 1.0-1.2 initially. The rate of decrease should be between 0.1 and 0.3/min. Any decrease
observed in the negative controls should be subtracted from the experimental values.
Convert A340 Change Rate to Enzyme Activity Concentration (mol/min/mL) and determine the total
activity (mol/min)
LuxG Exercise 4 Flavin Reductase Activity Assays
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 103
Fall 2018
Data Analysis: Specific Activity, Turnover Number and Purification Summary Table
Also see: Bradford Assay for Protein Concentration (Handling the Data) in the LDH section.
Specific Activity
This is the amount of enzyme activity per unit mass of protein. If the preparation is a mixture of
proteins, the mass includes all the proteins, not just the enzyme whose activity is quantified using the
Bradford. Specific activity is typically expressed in: (mol substrate converted)/min/mg. During a
purification procedure, this value usually increases because proteins other than the relevant enzyme are
gradually eliminated. The specific activity reaches a maximum level when the enzyme being quantified is
pure. For this LuxG-his6 preparation, the calculation will only be done for the affinity-purified sample.
(enz activ conc in mol/min/mL)*(conc prot in mg/mL)-1 = specific activity in mol/min/mg
Turnover Number
This is the number of substrate molecules (or moles) converted to product per active site (or mole of
active sites) every second. Units are simply s-1. It is also referred to as k2 or k-catalysis (kcat). Many
enzymes are multimers of two or more subunits and in some cases the multimer includes both catalytic
and regulatory subunits. In most cases there is a single active site in each catalytic subunit.
1)
2)
Calculate a turnover number for the affinity purified LuxG-his6 protein.
Compare the value obtained to those reported in the literature for LuxG and other flavin
reductases. Suggest explanations for significant differences.
Purification Summary Table
Complete the table shown below.
Step
Volume
(mL)
Mass
(mg)
Protein conc.
(mg/mL)
Total activity
(mol*min-1)
Activity conc.
(mol*min-1 *mL-1)
Specific activity
(mol*min-1 *mg-1)
Crude
extract
Affinity
purified
References
Berg, JM, Tymoczko, JL & Stryer, L (2007) The Michaelis-Menten Equation Describes the Kinetic
Properties of Many Enzymes. In: Biochemistry 6th ed, Freeman Press, pp 216-225.
Ingelman, M et al. (1999) Crystal Structure of NAD(P)H:Flavin Oxidoreductase from Escherichia coli.
Biochemistry 38: 7040-7049.
Nijvipakul, S et al. (2008) LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence
LuxG Exercise 4 Flavin Reductase Activity Assays
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
J Bacteriology, 190: 1531–1538
Voet, D and Voet, JG (2004) Enzyme Kinetics. In: Biochemistry, 3rd ed, Wiley Press, pp 477-482.
LuxG Exercise 4 Flavin Reductase Activity Assays
Page 104
Fall 2018
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 105
Fall 2018
Appendix
Derivation of Lineweaver-Burk equations for competitive, noncompetitive,
and uncompetitive inhibition
Competitive Inhibition
• 𝑣 = 𝑘2 𝐸𝑆
•
𝐸𝑇𝑜𝑡𝑎𝑙 = 𝐸 + 𝐸𝑆 + 𝐸𝐼
𝑘 𝐸𝑆
𝑣
= 𝐸 + 2𝐸𝑆 + 𝐸𝐼
•
𝐸
𝑇𝑜𝑡𝑎𝑙
•
•
•
•
𝑉𝑚𝑎𝑥 𝐸𝑆
𝐸 + 𝐸𝑆 + 𝐸𝐼
𝐸 𝑆
𝐸 𝑆
𝐿𝑒𝑡 𝐾𝑀 =
, 𝐸𝑆 =
𝐸𝑆
𝐾
𝐸 𝐼
𝐸 𝐼 𝑀
𝐿𝑒𝑡𝐾𝐼 =
, 𝐸𝐼 =
𝐸𝐼
𝐾𝐼
𝑣=
𝐸 𝑆
𝑣
𝑉𝑚𝑎𝑥
•
𝑣
𝑉𝑚𝑎𝑥
•
𝑣
=
𝐾𝑀
𝐸 𝑆
𝐸 𝐼
𝐸+𝐾 + 𝐾
𝑀
𝐼
𝐸
=
𝐸
𝑆
𝐾𝑀
𝐾 𝐼
(𝐾𝑀 + 𝑆 + 𝑀 )
𝐾𝑀
•
𝑉𝑚𝑎𝑥
𝑣=
•
1
•
1
𝑣
=
•
•
1
𝑣
=
𝑣
=
𝐾
=
𝐾𝐼
𝑆
𝐾 𝐼
(𝐾 + 𝑀 + 𝑆 )
𝑀
𝐾𝐼
𝑉𝑚𝑎𝑥 𝑆
𝐼
𝐾𝑀 (1+ )+ 𝑆
𝐾𝐼
𝐼
𝐾𝐼
𝐾𝑀(1+ )+ 𝑆
𝑉𝑚𝑎𝑥 𝑆
𝐼
𝐾 (1+ )
𝑀
𝐾𝐼
𝑉𝑚𝑎𝑥
𝐾𝑀 𝑎𝑝𝑝 1
𝑉𝑚𝑎𝑥 𝑆
𝑀𝑎𝑝𝑝
1
𝑆
+
1
𝑉𝑚𝑎𝑥
1
+ 𝑉𝑚𝑎𝑥
𝐼
= 𝐾 (1 + ) = apparent value of K
𝑀
𝐾𝐼
in the presence of inhibitor
M
Derivation of Lineweaver-Burk equations for competitive, noncompetitive, and uncompetitive inhibition
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 106
Fall 2018
Noncompetitive Inhibition
• 𝑣 = 𝑘2 𝐸𝑆
•
𝐸𝑇𝑜𝑡𝑎𝑙 = 𝐸 + 𝐸𝑆 + 𝐸𝐼 + 𝐸𝑆𝐼
•
•
•
•
•
𝑣
𝐸𝑇𝑜𝑡𝑎𝑙
𝑉𝑚𝑎𝑥 𝐸𝑆
𝐸 + 𝐸𝑆 + 𝐸𝐼 + 𝐸𝑆𝐼
𝐸 𝑆
𝐿𝑒𝑡 𝐾𝑀 =
, 𝐸𝑆
𝐸𝑆
𝐸 𝐼
𝐿𝑒𝑡𝐾𝐼 =
𝐸𝑆 𝐼
𝐸𝐼 = 𝐸𝑆𝐼
𝐸 𝑆
𝑣
𝑣=
=
𝑉𝑚𝑎𝑥
•
•
𝑘2 𝐸𝑆
𝐸 + 𝐸𝑆 + 𝐸𝐼 + 𝐸𝑆𝐼
=
=
=
•
𝐾𝑀
𝐸 𝑆 𝐼
𝐾𝑀 𝐸𝑆𝐼
𝐾𝑀
𝐸+
𝑣
𝐸 𝑆
𝐾𝑀
+
𝐸 𝐼
𝐾𝐼
𝐸
𝐸 𝑆 𝐼
+
𝐾 𝑀𝐾 𝐼
𝑆
𝐾𝑀
𝐸
𝐾 𝐼
𝑆 𝐼
(𝐾𝑀 + 𝑆 + 𝑀 +
𝐾𝑀
𝐾𝐼
𝐾𝐼
𝑉𝑚𝑎𝑥
=
𝑣
𝑉𝑚𝑎𝑥
= (𝐾𝑀+𝐾𝑀 𝐼 + 𝑆 + 𝑆
𝑆
𝐾𝐼
•
𝐸 𝑆
𝑉
𝐼
)
)
𝐾𝐼
𝑆
𝐼
𝑣 = 𝐾𝑀(1+ 𝐼𝑚𝑎𝑥
)+ 𝑆 (1+ )
1
𝑣
𝐾𝐼
𝐼
𝐾
𝐼
𝐾𝐼
𝐼
𝐾
𝐼
𝐾𝑀(1+ )+ 𝑆 (1+ )
=
𝑉𝑚𝑎𝑥 𝑆
𝐼
𝐾𝑀(1+ ) 1
1
𝐾𝐼
𝑉𝑚𝑎𝑥
•
𝑣
=
•
1
𝑣
=𝑉
•
𝑉𝑚𝑎𝑥𝑎𝑝𝑝 =
𝐾𝑀
𝑚𝑎𝑥𝑎𝑝𝑝
𝑆
1
𝑆
1+
+
𝐼
𝐾𝐼
𝑉𝑚𝑎𝑥
1
𝑉𝑚𝑎𝑥
1+ 𝐼
+ 𝑉𝑚𝑎𝑥
𝑎𝑝𝑝
= apparent value of Vmax in the presence of inhibitor
𝐾𝐼
Uncompetitive Inhibition
• 𝑣 = 𝑘2 𝐸𝑆
•
𝐸𝑇𝑜𝑡𝑎𝑙 = 𝐸 + 𝐸𝑆 + 𝐸𝑆𝐼
𝑘2 𝐸𝑆
𝑣
•
𝐸𝑇𝑜𝑡𝑎𝑙 = 𝐸 + 𝐸𝑆 + 𝐸𝑆𝐼
•
•
•
•
𝑉𝑚𝑎𝑥 𝐸𝑆
𝐸 + 𝐸𝑆 + 𝐸𝑆𝐼
𝐸 𝑆
𝐿𝑒𝑡 𝐾𝑀 =
𝐸𝑆
𝐸𝑆 𝐼
𝐿𝑒𝑡𝐾𝐼 =
𝐸 𝑆 𝐼
𝐸𝑆𝐼 = 𝐾𝑀 𝐸𝑆𝐼
𝑣=
𝑣
𝑉𝑚𝑎𝑥
𝐸 𝑆
=
𝐾𝑀
𝐸 𝑆
𝐸 𝑆 𝐼
𝐸+
+
𝐾𝑀
𝐾 𝑀𝐾 𝐼
=
𝐾𝑀
𝑆 𝐼
(𝐾𝑀 + 𝑆 +
𝐾𝑀
𝐾𝐼
𝐸
•
•
𝑣
𝑉𝑚𝑎𝑥
𝑣
𝑉𝑚𝑎𝑥
𝑆
𝐸
= (𝐾
)
𝑆
+𝑆+
𝑀
𝑆 𝐼
)
𝐾𝐼
Derivation of Lineweaver-Burk equations for competitive, noncompetitive, and uncompetitive inhibition
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
•
•
•
•
•
𝑣=𝐾
𝑀
1
𝑣
1
𝑣
1
𝑣
=
=
𝑉𝑚𝑎𝑥 𝑆
𝐼
+ 𝑆 (1+ )
𝐾𝐼
𝐼
𝐾𝐼
𝐾𝑀 + 𝑆 (1+ )
𝑉𝑚𝑎𝑥 𝑆
𝐼
𝐾𝑀 1
𝐾
+ 1+ 𝐼
𝑉𝑚𝑎𝑥
𝑉𝑚𝑎𝑥 𝑆
𝐾
𝑀
= 𝑉𝑚𝑎𝑥
𝐾𝑀
𝑉𝑚𝑎𝑥
Page 107
Fall 2018
=
1
𝑆
1
+ 𝑉𝑚𝑎𝑥
𝐾𝑀𝑎𝑝𝑝
𝑉𝑚𝑎𝑥𝑎𝑝𝑝
𝑎𝑝𝑝
𝐼
𝐾𝑀(1+ )
=
𝐾𝐼
𝐼
𝑉𝑚𝑎𝑥(1+ )
𝐾𝐼
Derivation of Lineweaver-Burk equations for competitive, noncompetitive, and uncompetitive inhibition
Reagents for Polyacrylamide Gel Electrophoresis (PAGE)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
30:0.8 Acrylamide/Bis-acrylamide (store at 4 C).
a.
30% Acrylamide.
b.
0.8% Bis-acrylamide.
Tris hydrochloride buffer for native separating gel (4X).
a.
24.0 mL of 1.000 M HCl.
b.
18.15 g Tris base.
c.
Water to 100 mL.
d.
Measure pH but do not adjust. The pH should be about 8.8.
Tris hydrochloride buffer for native stacking gel (4X).
a.
3.03 g Tris base.
b.
20 mL of water.
c.
Adjust to pH 6.8 with 1 M HCl (24 mL).
d.
Bring to 50.0 mL.
Tris glycine running buffer for native gels (10X).
a.
30.25 g Tris base.
b.
144 g glycine.
c.
Water to 1.00 L.
d.
Measure pH but do not adjust. The pH should be about 8.6.
4X Buffer for the SDS separating gel (filtered, store at room temperature).
a.
0.4% Sodium dodecyl sulfate.
b.
1.5 M Tris.
c.
Hydrochloric acid to pH 8.8.
4X Buffer for the SDS stacking gel (store at room temperature).
a.
0.4% Sodium dodecyl sulfate.
b.
0.5 M Tris.
c.
Hydrochloric acid to pH 6.8.
10X Tris-glycine running buffer for the SDS gels (filtered, store at room temperature).
a.
1.0% Sodium dodecyl sulfate.
b.
0.25 M Tris.
c.
1.92 M Glycine.
d.
Measure pH, but do not adjust. The pH should be about 8.6.
TEMED (store at 4 C).
10% Ammonium persulfate (made fresh daily).
40% Sucrose, 0.25% bromophenol blue.
Persulfate-riboflavin solution (4X; made fresh each day).
a.
b.
c.
60 mg K2S2O8.
2 mg riboflavin.
Water to 100 mL
Reagents for Polyacrylamide Gel Electrophoresis (PAGE)
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 110
Fall 2018
Operation of the Spectronic 20D+ (Spec 20)
Although you will be using the Spec 20 for absorbance measurements, it is best to initially set the
baseline in transmittance mode. After achieving a 100% transmittance reading with a blank (water or
the buffer that will be used for the assay), switch to absorbance mode. The reading should be close to
zero.
1) Set the Baseline in Transmittance Mode
The spectrophotometer is turned on by rotating the amplifier control (left-hand knob) clockwise. This
should be done at least 20 min before measurements are made. Set the mode switch to “transmittance”
and select the appropriate wavelength. After the instrument has warmed up, adjust the amplifier
control knob so that the display reads 0 on the percentage transmission scale when no light is striking
the phototube (no tube in holder).
The right-hand (light control) knob regulates the amount of light passing through the second slit to the
phototube. This light control knob is needed because the light source emits light of different
wavelengths at different intensities and the phototube is not equally responsive to light of different
wavelengths. Besides this, the "blank" solution used to zero the instrument, usually the solvent in which
the light-absorbing species, or "chromophore", is dissolved, may itself absorb light of certain
wavelengths. In order to measure the absorbance due to only the chromophore, this background
absorbance must be compensated for. Therefore, after the spectrophotometer has been zeroed when
no light is hitting the photocell, a blank solution is placed in the light path and the light control knob is
rotated until the display reads 100% T. Both this blank solution and all of the samples you place in the
spectrophotometer must have volumes greater than or equal to 3 mL. This adjustment achieves the
desired compensation. If a sample solution is now placed in the light path, any change in the percent
transmittance is due to only the particular chromophore in the sample and the percent transmittance is
related to the concentration of that chromophore.
Whenever a change in wavelength is made, the zero transmittance and 100% transmittance must be
reset, since the amount of compensation needed varies with wavelength. The display reading should go
to 0% transmittance whenever the cuvette is removed from the sample holder, because removing the
cuvette releases an occluder that drops into the light beam and prevents the beam from reaching the
phototube. Adjusting the zero transmittance on the scale can therefore always be done simply by
removing the cuvette.
2) Switch to Absorbance Mode to Get Data
With the blank sample still in the holder, switch to absorbance mode. The reading on the display should
now be close to zero. Bring it to exactly zero with the light control knob. You may now start assaying
experimental samples.
Properties of the Phototube
The phototube is a photoemissive cell of the cesium-antimony surface type. The relative response of the
phototube to a beam of monochromatic light of constant intensity is as follows:
Operation of the Spectronic 20D+ (Spec 20)
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Wavelength
(nm)
350
400
450
500
550
600
625
Page 111
Fall 2018
Response
(Relative to 400 nm)
0.90
1
0.91
0.68
0.37
0.10
0.05
It can be seen that the phototube is much more sensitive to light of wavelength 400 nm than to light of
wavelength 600 nm. This means that the phototube will require a greater influx of monochromatic light
at 600 nm than at 400 nm in order for the same transmittance reading to be registered upon the
colorimeter dial.
We usually use disposable glass culture tubes with the Spec 20 rather than “cuvettes”. However some of
the discussion below is still relevant.
Use of Cuvettes with the Spectronic 20
The handling of the cuvettes is extremely important. Often two cuvettes are used interchangeably: one
for the blank solution and one for the samples to be measured. Any variation in the cuvette, however,
such as a change in the width of the cuvette or the curvature of the glass or stains, smudges or
scratches, will cause the results to vary. Thus, it is essential in handling the cuvettes to follow several
important rules:
Do not touch the lower portion of a cuvette through which the light beam will pass.
If possible, always rinse the cuvette with several portions of the solution before taking a measurement.
With precious samples of protein, rinse with the buffer.
Wipe off any liquid drops or smudges on the lower half of the cuvette with a clean Kimwipe before
placing the cuvette in the instrument. NEVER wipe cuvettes with towels or handkerchiefs.
To avoid any possible scratching of the cuvette in the optical path, insert the cuvette into the sample
holder with the index line facing toward the front of the instrument.
After the cuvette is seated, line up the index lines exactly.
The cuvette should be removed in the reverse manner.
When using two cuvettes, always use one of the cuvettes for the "blank" solution and always use the
other cuvette for the various samples being measured. The two cuvettes must be checked to make sure
that they give the same blank values.
Operation of the Spectronic 20D+ (Spec 20)
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 112
Fall 2018
Preparation of an Acrylamide Gel for Electrophoresis
Do all of the assembly described below before mixing the gel solution. Once the polymerization catalysts
are added, the solution must be immediately poured into the assembly.
Assemble plates and spacers before mixing gel solution.
Desired arrangement after both parts of gel are formed.
Use of the JGC System for Gel Casting
Adapted from user’s guide: Thermo Scientific Owl Separations Systems www.owlsci.com
Components: Casting stand with two thumbscrews and clear acrylic Spacer Plate. White Spacer Placer
Card (slightly narrower than glass gel plates). Plastic pouch (slightly wider than glass gel plates).
Plastic wash bottle with 95% ethanol.
Arrange the two side spacers between two glass plates. A bottom spacer is NOT required. The pouch
holds the solution between the plates
Insert well-forming comb between plates. Mark on the notched plate (black Sharpie pen) the desired
position of top edge of separating gel (~1 cm below bottom edge of comb). Remove comb.
Preparation of an Acrylamide Gel for Electrophoresis
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 113
Fall 2018
Place the plates and spacers assembly into a plastic gel pouch.
The gel solution will be poured from a beaker or flask (NOT pipetted) between the plates. In
some cases the separating gel will be prepared a day or more before use and must be stored in a
manner that prevents loss of moisture. Trim the top of the bag with a razor so that pouring the gel
solution in will be easy, but the bag extends far enough above the plates so that it may be folded over to
prevent loss of moisture during storage.
5) Stand the glass and bag assembly up in the casting stand so that it rests against the rear wall. Use the
Spacer Placer Card
to align the spacers by sliding the card between the glass plates. Spacers are automatically aligned on
either side of the glass plate.
6) Insert the clear acrylic Spacer Plate in front of the glass and bag assembly. Be sure to place this spacer
plate IN FRONT of the glass plate/bag assembly; i.e. the acrylic should contact the thumbscrews, not the
glass.
Tighten the two thumbscrews at the front of the unit just enough to hold the glass plate/bag assembly
snug between the Spacer Plate and the rear wall of the casting stand.
Use the thumbscrews on the base of the unit to level and stabilize the casting stand.
Immediately after adding polymerization catalysts, pour the separating gel mixture between the
plates. Bring the level up to the mark made earlier for the desired position of the top, then overlay
with 95% ethanol. The overlay excludes O2 which can inhibit polymerization.
Allow 20 min for polymerization. Instructions for preparing protein samples and the
stacking gel are in individual exercises.
Preparation of an Acrylamide Gel for Electrophoresis
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 114
Fall 2018
Absorbance Measurements with Multiwell Plates and an Automated Plate
Reader
An alternative to using a conventional spectrophotometer for assaying UV or visible light absorbance is a
plate reader. Plate readers offer several advantages:
A smaller volume of each sample (200 L) is used. Conventional spectrophotometry typically requires
0.5-3 mL for each measurement. Thus, less of a precious protein sample need be sacrificed for the
measurement. This also conserves on staining reagents such as that used for the Bradford assay.
Ninety-six samples may be assayed in a multiwell plate. This is less expensive than buying disposable
tubes for Spec 20 measurements.
Fewer manipulations are necessary.
Choose Appropriate Type of Plate
Multiwell plates are made from a variety of types of plastic. For visible light absorbance, standard plastic
works best. However for wavelengths less than 400 nm, a special UV-transparent plastic is used. Be
certain to use the standard plastic for the Bradford assay (595 nm) and the UV-transparent for the A280
assay (the A595 signal is dampened by the UV-transparent plastic.) Wells must have a flat-bottom for
consistent readings.
Nunc 269620: standard plastic, 96 flat-bottom wells.
Costar 3635*: UV-transparent, 96 flat-bottom wells.
*These are more expensive than the Nunc plates so we reuse them. Just rinse with dH2O before the
sample dries in the wells.
Bradford Assay for LuxG and LDH Exercises (total reaction vol per well = 200 L)
The standard curve (with -globulin) should extend to 30 g/mL, giving a maximum A595 of 0.4-0.5. Use a
0.1 mg/mL stock and appropriate volumes of staining reagent to prepare reactions with 0, 1, 2, 3, 4, 5
and 6 g protein.
The A595 reading of the sample being analyzed is only useful if it is within the range of the standards, so
estimate aliquot volumes that will contain between 1 and 6 g. For the LuxG exercises: assay 10 L for
affinity-column and desalting column fractions. For LDH Exercise 7: assay 20 L of each diluted bovine
heart sample.
After mixing protein with stain, allow 20 min for the color to stabilize.
Preparing Samples and Operation of the Plate Reader
Absorbance Measurements with Multiwell Plates and an Automated Plate Reader
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 115
Fall 2018
It will be most practical if your TA operates the plate reader because there is only one machine and
many students. Each plate to be assayed should have at least one well containing 200 L of the
appropriate blank solution: staining reagent with no protein for the Bradford, or Tris/glycerol/DTT for
the A280. For each Bradford plate there should be at least one set of standard BSA reactions as described
above. Your TA will instruct you as to the "layout" of the samples in the wells. It is critical that the blank
sample be in the appropriate well on each plate because the analysis program will subtract this reading
from all the other readings. For the Bradford, the protocol in the control program may also be set to
label particular wells for the standards. Since each plate has 96 wells, samples from several pairs of
students will be loaded in each plate. Your TA will also help you to obtain a Table of the data in MS-Excel
and/or PDF format.
Absorbance Measurements with Multiwell Plates and an Automated Plate Reader
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 116
Fall 2018
Increasing the Concentration of Protein with a Centrifugation/Filtration
Device
(from the Millipore company 2009)
www.millipore.oom
2
Introduction, continued
The Amicon Ultra-4 product line includes 5 different cutoffs (Nominal Molecular Weight
Limit, NMWL, or Molecular Weight Cutoff, MWCO}:
Amicon Ultra 3K devic.e-3,000 NMWL
Amicon Ultra 10K device -10,000 NMWL
Amicon Ultra 30K device-30,000 NMWL
Amicon Ultra 50K device -50,000 NMWL
Amicon Ultra 1OOK device -100.000 NMWL
Amicon Ultra-410K filter devices are for In vitro diagnostic use and can be used to concentrate serum,
urine. cerebrospinal fluid, and other body fluids prior to analysis.
Amicon Ultra-4 3K,30K,SOK,and 100K filter devices are for research use only and not for use in
diagnostic procedures.
Applications
Concentration of biological samples containing antigens, antibodies, enzymes, nucleic acids (DNA/RNA
samples, either single- or double-stranded), microorganisms. column eluates. and purified samples
Amicon Ultra-4 Centrifugal Filler Devices 3
Applications, continued
Purification of macromolecular components found in tissue culture extracts and cell lysates. removal of
primer. linkers. or molecular labels from a reaction mix, and protein removal prior to HPLC
Desalting. Buffer exchange, or diafiltration
Materials Supplied
The Amicon UHra-4 device is supplied with a cap. a filter device. and a centrifuge tube.
Increasing the Concentration of Protein with a Centrifugation/Filtration Device
Techniques in Protein Chemistry
CHEM 108
Crowley, Davidson, Duffy, Johnson, Kyte & Lumba
Page 117
Fall 2018
How to Use Amicon Ultra-4 Centrifugal Filter Devices
1. Add up to 4 mL of sample {3.5 mL if using a 23' fixed angle rotor} to the Amicon Ultra filter
device.
2. Place capped filter device into centrifuge rotor {swi1gingbucketpreferred}: counterbalance with
a similar device.
3. When using a swinging bucket rotor: Spin the device at 4.000 x g maximum for approximately
10--40 minutes.
When using a 35°fixed angle rotor for AmIcon Ultra 3K,10K,30K,and 50K devices: Spin at 7.500 x
g maximum for approximately 10--40 minutes.
When using a 35°fixed angle rotor for Amicon Ultra 100K devices: Spin at 5,000 x g maximum for
approximately 10-20 minutes.
N0TE: Refer to Figures 1and 2,and Tables 3 and 4 or typical spin times.
4. To recover the concentrated solute, insert a pipette into the bottom of the filter device and
withdraw the sample using a side-to-side sweeping motion to ensure total recovery. The
ultrafiltrate can be stored in the centrifuge tube.
NOTE: For optimal recovery, remove concentrated sample immediately after centrifugation.
Increasing the Concentration of Protein with a Centrifugation/Filtration Device