39
Unit 3
Analysis of Proteins by Western Blots
Introduction:
Electrophoresis
Electrophoresis is the process of a biological sample into a molecular matrix and applying an electric field, providing
excellent resolution of complex mixtures commonly used as an analytical technique. Electrophoresis separates
charged molecules according to their size, shape and charge. Typically a gel made of either agarose or
polyacrylamide provides the support system for resolving molecules in an electric field. The macromolecules are
placed in a well at one end of the gel and an electrical current is applied through a buffer across the system. Gels
made from polymerized acrylamide (polyacrylamide gels) are frequently used for separating proteins and small-sized
nucleic acids because they have high resolving power, do not interact with biomolecules, and have a stable matrix.
Typically the polyacrylamide is crosslinked with N,N’-methylene-bis acrylamide in a n aqueous polymerization
reaction that is initiated by the molecule N,N,N’,N’-tetramethylethenediamine (TEMED). If low concentrations of
acrylamide and bis-acrylamide are used, the pores formed are larger, allowing for analysis of higher molecular weight
proteins. Conversely, smaller molecular weight molecules are analyzed in gels with higher concentration of
acrylamide and bis-acrylamide. The entire complex forms a matrix of fibers as shown in Fig. 1 below, where
represent the polyacrylamide and the
represent the crosslinking:
Fig. 1: Matrix of Polyacrylamide with Crosslinking
The PAGE gels are polymerized between two square panes of glass or plastic that are sealed around the edges and
are stood upright during polymerization. A comb is inserted at the top edge of a gel after pouring the polymerizing
reaction between the plates, in order to produce the wells into which the samples are placed. The resulting gels are
run upright as well, giving rise to the common name of “vertical gel electrophoresis”.
PAGE gels may be continuous or discontinuous. Continuous gels have the same concentration throughout the gel or
a gradient of acrylamide and bis-acrylamide concentration with the most concentrated gel at the bottom of the gel.
More often, a discontinuous gel is used. In this gel the lower 80 percent of the gel is called the resolving gel. This gel
usually contains a higher percentage of polyacrylamide/bis-acrylamide and is poured first. On top, there is a stacking
gel that contains a lover percentage of polyacrylamide/bis-acrylamide and prepared with a buffer containing a lower
concentration of buffer salts. The stacking gel has lower conductance than the resolving gel. This means that the
proteins will carry the current more and therefore travel quickly through the stacking gel until it reaches the resolving
gel, where they concentrate at the interface, in a “stack”. This allows the proteins to enter the resolving gel close
together and therefore increases the resolution during electrophoresis in the resolving gel, where the smaller pore
size exerts a stronger frictional drag on the proteins during their separation.
Native gels
Native gels are run on proteins when the native conformation of the protein needs to be preserved, usually because
the location of the protein of interest must be identified by its structural function. For example, if an enzyme must be
identified by its enzymatic activity, a native gel will be used. Also, Western blots are often performed with native gels
to ensure that the antibody detection of a protein of interest will work.
When an electrical field is applied to the proteins they will travel from the wells where they were loaded, down into the
acrylamide gel towards the positive electrode (the anode) IF the net charge of the protein is negative. While the
electric field accelerates negatively charged proteins towards the anode, their rates of movement is influenced by
frictional drag, in turn affected by factors such as size and shape of the molecule. Smaller proteins will move more
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
40
quickly through the gel than larger ones due to less frictional drag. For the same reason, tightly coiled globular
proteins will travel more quickly than more loosely packed proteins or fibrous proteins having a more extended
structure in an electrophoretic gel.
The ratio of charged amino acids in a protein influences movement through the gel by determining the net charge on
the protein; those proteins with more negatively-charge (acidic) amino acids than positively-charged (basic) amino
acids will travel more quickly towards to positive anode due to their large net negative charge. A protein with a net
positive charge will, in fact, migrate the wrong direction from the sample well, away from the anode and towards the
cathode. The net charge of a protein can be manipulated by a change in pH. Buffers at lower ranges of pH will shift
the dissociation of acidic amino acid side groups (aspartate and glutamate) towards their protonated, or uncharged
state. At the same time these acidic buffers will shift the basic amino acid side groups (arginine, lysine, and histidine)
towards their protonated, or positively charged state. If basic buffering systems are used, the acidic side groups will
become deprotonated and acquire a negative charge, while the basic side groups will shift towards their
deprotonated, or uncharged state. For that reason, most native gel buffering systems operate in a pH range of 8-9,
where all but the most basic of proteins will carry a net negative charge and will migrate into the gel towards the
anode.
The rate of migration in a native gel is not strictly a function of size, but is influenced by the size-to-mass ration of the
proteins. Highly acidic proteins will have a higher negative charge per unit mass than a more basic protein, so will
migrate faster during electrophoresis regardless of size differences between the two proteins.
Denaturing gels (SDS-PAGE)
With all these factors influencing the movement of native proteins through the PAGE (polyacrylamide gel
electrophoresis) gel, it is nearly impossible to predict where a given protein will migrate or to analyze the relative size
of proteins separated on a native gel. In order for proteins to behave similarly in a electric field, their differences in
charge-to-mass ratio and their difference in shape must be made to be uniform. This uniformity is accomplished by
denaturation of proteins into a uniformly extended shape and coating of proteins with negative charges. That way the
unfolded proteins will be accelerated uniformly by the electric field and the frictional drag through the gel matrix will be
a simple function of size.
The denaturation and coating of protein molecules with negative charges is accomplished by the use of heat and an
ionic detergent called sodium dodecyl sulfate (SDS). This detergent is the sodium salt of a 12-carbon alkyl sulfate
compound. In combination with heat, it disrupts the secondary, tertiary, and quaternary structures of the protein by
breaking the ionic and hydrogen interactions between the amino acids of the protein, as well as interfering with the
hydrophobic interactions responsible for correct folding of the protein. Once denatured, SDS will coat the denatured
proteins’ hydrophobic amino acid side groups with its hydrophobic dodecyl tail, thereby coating the protein with
negative charges from the sulfate head group of the detergent. As long as SDS remains in solution with a denatured
protein, it will not renature.
Reducing agents are often added to SDS in order to break any disulfide bonding that holds tertiary and quaternary
proteins structures in a protein molecule. Dithiothreitol and 2-mercaptoethanol are short molecules containing a
sulfhydryl (-SH) group that will convert the disulfide cystine bridges into two cysteine side groups to release any
tertiary structure and/or quaternary structure of the proteins due to disulfide bridges. (The sulfhydryl group is what
gives these compounds their “rotten egg” smell.)
Typically, 1-2% SDS and 0.1 M mercaptoethanol buffered at pH 6.8 are used along with high temperatures to
completely denature proteins and coat their extended structures with negative charges. In addition to the
mercaptoethanol and SDS denaturants, the sample buffer contains a tracking dye that will travel with the front and
determine how far the gel has run, Glycerol is also included for increased density of the sample in order for it to settle
into the bottom of the well and not go floating off into the electrophoresis buffer. Typically somewhere between 10
and 40 g protein are loaded into a well, depending on the purity of proteins in the sample. This total volume is
typically about 20-40 L, half of which is protein and half is the 2X sample buffer.
Gels are generally run in a Tris-glycine buffer system with 0.1% SDS, pH 8.9. When the tracking dye from the
sample buffer reaches the end of the resolving gel, proteins can be fixed into place (preventing diffusion of the
separated bands) by acidified methanol solutions. The protein bands can be analyzed directly following a staining
procedure with Coomassie blue dye. This gives rise to the SDS-PAGE electrophoresis that is the most common
method of identifying an unknown protein or determining its molecular weight. A set of proteins markers of known
molecular weight are run on the same gel along with the unknown. Analysis of sizes of protein bands in the gel is
then a straightforward comparison to the migration distances relative to the molecular weight markers.
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
41
Blotting Techniques
All blotting techniques use the same principle: macromolecules are transferred out of a gel onto another matrix, either
nylon or nitrocellulose. This matrix may then be queried by a probe to visualize the bands of molecules. For
example, in Southern blotting, DNA is subjected to electrophoresis and then a nylon sheet is used as the matrix to
“blot” the gel. The nylon is treated with radioactively-labeled DNA probe molecules that will bind to specific bands on
the membrane. The resulting DNA is then visualized by autoradiography. In a Western blot procedure, a specific
protein separated on an electrophoretic gel can be identified by an immunological staining procedure. The
separated proteins must be prepared for antibody binding by transfer to a membrane by electroelution, the “blotting”
technique.
The following table summarizes the kinds of blotting that are typically done and the probes used.
Molecules to be identified
Probe
Blotting system
Southern
Northern
Western
South-Western
DNA
RNA
(usually mRNA)
Protein
protein
(which can bind to DNA)
Radiolabelled
DNA
Radiolabelled
DNA
Antibody
dsDNA
The steps of the Western blotting procedure for analysis of proteins is listed below:
1) Electrophoresis. Proteins are separated by gel electrophoresis, usually by SDS-PAGE using a
tris/glycine/SDS running buffer.
2) Transfer. The proteins are transferred to a sheet of special membrane, usually nitrocellulose
polyvinyl pyrolidon, or nylon, though other types of paper, or membranes, can be used.. This
transfer is generally done by electroelution at 90 degrees to the gel through a buffer. The proteins
retain the same pattern of separation they had on the gel. The blotting buffer is generally similar to
the electrophoresis buffer, but lacking SDS. An alcohol (methanol, ethanol, or isopropanol) is
added to the blotting buffer to facilitate protein binding to the membrane.
3) Protein Visualization.
a) Blocking. The blot is incubated with a generic protein (such as nonfat milk proteins) and a
nonionic detergent such as Tween 20 to bind to any remaining sticky places on the nitrocellulose.
This prevents nonspecific binding of antibody to the membrane.
b) Primary Antibody. An antibody (monoclonal or polyclonal) directed against the protein of interest
is then incubated with the gel to allow it to bind to its specific protein. The antibody is diluted in a
buffer solution, generally phosphate-buffered saline (PBS) containing a carrier protein, generally
bovine serum albumin (BSA), along with some nonionic detergent such as Tween 20. The
additives to the buffer help to ensure that the antibody is specific for the protein of interest, and
does not bind to other proteins on the membrane.
c) Secondary Antibody. Since the antigen-antibody complexes are not colored, they must be
treated in some way in order to visualize them. Usually an enzyme such as a horseradish
peroxidase or alkaline phosphatase is coupled to a secondary antibody that binds to
immunoglobulin (Ig) chains of the primary antibody. Alternatives to the conjugated enzyme are
radiolabelling or dye conjugation of the secondary antibody.
d) Developing. The unbound secondary antibodies are washed away, and the conjugated enzyme is
then presented with a colorless substrate which when reacted, will produce a colored product. The
appearance of a colored enzyme product indicates the location on the gel of the protein of interest.
Band densities in different lanes can be compared providing information on relative abundance of
the target protein. Thus, the Western blot is similar to an ELISA. The major difference is the
resolution and transfer to a membrane of the protein before being presented with antibodies.
The table below lists the enzymes and substrates commonly used for detection of target proteins in
a Western blot.
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
42
Enzyme
Substrate
Color of reaction product
Alkaline phosphatase
5-bromo-4-chloro-3’-indolylphosphate with nitroblue
tetrazolium chloride (BCIP/NBT)
Black/purple
naphthol AS-MX phosphate with Fast Red TR
Bright red
4-chloronaphthol (4-CN)
Blue/purple
3-amino-9-ethyl carbazole (AEC)
Red/brown
3,3’-diaminobenzidene tetrahydrochloride (DAB)
(note: carcinogenic!)
Brown
Phenazine methosulfate with nitroblue tetrazolium
chloride
Black/purple
Horseradish peroxidase
Glucose oxidase
Troubleshooting and optimizing a Western blot
Like all complex procedures, there are many technical difficulties that can lead to disastrous results. Careful attention
to the correct temperatures and incubation times is very important to the success of a Western blot, as is the quality
of the reagents being used. The blotting step itself is critical: it is important to layer the gel onto the onto a prewetted
membrane with good contact and without any air bubbles. The elution time must be optimized for the size of protein
of interest. Overheating during electroelution must be carefully avoided for efficient transfer of proteins to a
membrane. The following guide can help to be used to optimize a Western blot procedure for a specific application,
and to determine exactly which step of the protocol may be a problem when getting disappointing results.
Problems associated with the electrotransfer blotting stage:
Symptom
Possible cause
Remedy
Band smeared/distorted
Weak signal
Membrane not uniformly
wetted prior to transfer.
Many types of membranes are hydrophobic, and
must be prewetted with methanol; the entire
membrane should change uniformly from opaque to
semi-transparent.
Air bubbles under membrane
and between other layers in
the stack.
Using a pipet or stirring rod, gently roll out any
trapped air bubbles while assembling the stack.
Uneven contact between gel
and membrane
Make sure the entire gel and membrane surfaces are
in good contact.
Too much heat generated
during the transfer
Pre-chill the buffer, carry out the transfer in a cold
room, or reduce the current.
Proteins transferred too
rapidly; protein buildup on the
membrane surface
Reduce the strength of the electrical field.
Incomplete transfer of proteins
Stain the gel after the transfer to check for residual
proteins. If transfer was not complete, review your
transfer technique. Improper buffer concentrations,
or too much methanol, for example, can lead to poor
transfer efficiency.
Proteins passing through the
membrane.
Increase the time the proteins have to interact with
membrane during the transfer by reducing the voltage
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
43
by as much as 50%.
Highly negatively charged proteins tend to move
quickly in an electric field. Decrease the voltage to
slow down migration of these proteins
The presence of SDS in the gel may inhibit protein
binding. Equilibrate the gel in the transfer buffer for at
least 15 minutes prior to the transfer.
Methanol concentration in transfer buffer is too low to
facilitate removal of SDS. Increase the methanol to
15-20%, especially for smaller molecular weight
proteins.
Inadequate prewetting of the membrane will impede
binding of proteins.
Proteins retained in the gel.
If the methanol concentration in the transfer buffer is
too high, it can remove SDS from proteins and lead to
protein precipitation in the gel, reducing the transfer
of large proteins out of the gel. If this is an issue, the
transfer buffer can be supplemented with SDS
(0.01%-0.05%) or methanol concentration can be
reduced.
Isoelectric point of the protein
is at or close to the pH of the
transfer buffer.
A protein has no net charge at a pH that matches its
isoelectric point, so will not migrate in an electric field.
Try a higher pH buffer such as 10mM CAPS buffer at
pH 11, including 10% methanol.
Poor protein retention on
membrane
Once the transfer is complete, be sure to dry the
membrane completely to obtain optimal binding and
fixation of the proteins. This should be done prior to
any downstream detection method.
No signal
No transfer of proteins
Check for the gel and membrane orientation during
the transfer process. Use pre-stained molecular
weight standards to monitor the transfer.
Poor transfer of small
sized proteins (<10kDa)
SDS interferes with binding of
small molecular weight
proteins.
Remove SDS from the transfer solution.
Low methanol concentration in
the transfer buffer.
Use higher % methanol (15-20%) in the transfer
buffer.
Insufficient protein binding
time.
A lower voltage may optimize binding of small
proteins to the membrane.
Current doesn’t pass through
the membrane.
Cut the membrane and blotting paper exactly to the
gel size; do not allow overhangs.
Methanol concentration is too
high.
Reducing the methanol concentration to 10% (v/v) or
less should help in the transfer process by allowing
the gel to swell and reducing SDS loss, thereby
reducing the protein precipitation in the gel. Larger
proteins may require a longer transfer duration or a
higher current setting.
Poor transfer of large
sized proteins (>80 kDa)
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
44
Poor transfer of a wide
range of protein sizes.
Different conditions required to
transfer large and small
proteins
Try this protocol: “Transfer of a brad MW range of
proteins may require a multi-step transfer” by t. Otter
et al, Anal. Biochem. 162:370-377 (1987)
Problems associated with the protein visualization stage:
Symptom
Possible cause
Remedy
Weak or uneven stain
Not enough protein loaded in
the electrophoretic gel
Make sure you are loading the right amount of protein
in the electrophoresis gel by protein concentration
analysis prior to loading or staining for proteins in a
gel after a run.
Low levels of target protein in
the protein loaded onto the
electrophoretic gel.
The resolution of SDS-PAGE is 40-100 bands. If the
relative concentration of the antigen of interest is too
low (less than 0.2% of total protein) and not resolved
from another protein on the gel, the other protein can
block the binding of antibody. Enrichment of the
antigen by a purification step should be considered.
Membrane wasn’t adequately
prewetted in methanol.
Membranes are hydrophobic, and must be prewetted
with methanol; the entire membrane should change
uniformly from opaque to semi-transparent.
Improper blocking reagent
The blocking agent may have an affinity fro the
protein of interest and thus obscure the protein from
detection. Try a different blocking agent and/or
reduce both the amount or exposure time of the
blocking agent.
Insufficient antibody binding.
Increase the incubation time.
Multiple freeze-thaw or bacterial contamination of
antibody solution can change antibody titer or activity.
Increase antibody concentration or prepare it fresh.
Use fresh substrate and store properly. Outdated
substrate can reduce sensitivity.
Uneven/splotchy results
Poor transfer efficiency
Optimize protein transfer (see above)
Dried blot in membrane
Try rewetting the blot in water to maximize the
reaction.
Poor water quality
Water contaminants can interfere—use high quality
reverse osmosis/deionized water.
Denaturation destroys epitope
Separate proteins in a non-denaturing (native) gel
prior to the blotting.
Insufficient volume of staining
solutions
Use sufficient volumes of incubation solutions and
ensure that the membrane is completely covered with
these solutions during incubation. The container
used should be large enough to allow solution to
move freely across the blot. Do not incubate more
than one blot at a time in that same container. In
addition, the protein side of the blot should be facing
up so as not to be interaction with the bottom surface
of the container.
Air bubbles
The blot should not have any air bubbles on the
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
45
surface. Gently pull the membrane across the edge
of the container to remove bubbles.
Poor reagent quality
All of the buffers and reagent should be fresh and
free of particulates and contaminants. Micropore
filtration (0.2-0.45 um) of solutions and centrifugation
of antibody stocks may be required.
Fingerprints, fold marks, or
forceps imprints on the blot.
Avoid touching or folding membranes; use gloves
and blunt end forceps to handle them.
Nonspecific protein binding to
the membrane
Make sure to use clean electrotransfer equipment
and components and high quality reagents and water.
Insufficient washes
Increase washing volumes and durations. Prefilter all
of your solutions including the transfer buffer using
micropore filters (0.2-0.45 um). A stronger detergent
such as NP-40 or SDS may provide a more stringent
wash.
Secondary enzyme-conjugated
antibody concentration too
high
Increase antibody dilution
Nonspecific protein
interactions
Use Tween-20 (0.05%) detergent in the wash and
detection solutions to minimize protein-protein
interactions.
Poor reagent quality
Water contaminants can interfere—use high quality
reverse osmosis/deionized water.
Crossreactivity between
blocking reagent and antibody
Use different blocking agent or use Tween-20
(0.05%) detergent in the washing buffer.
Membrane drying during
incubation process
Use volumes sufficient to cover the membrane during
incubation
Speckled background
Aggregates in the blocking
reagent or secondary antibody
solution
Filter the blocking reagent and secondary antibody
solutions though micropore filters (0.2-0.45 um).
Nonspecific binding of
antibodies to other
proteins (multiple or
“ghost” bands)
Primary antibody concentration
too high.
Decrease concentration
Secondary antibody
concentration too high.
Decrease concentration
Antigen concentrations too
high.
Make sure you are loading the right amount of protein
in the electrophoresis gel by protein concentration
analysis prior to loading or staining for proteins in a
gel after a run.
Proteolytic breakdown of
antigen
If samples are stored for prolonged time or are not
stored properly, may result in a “family” of antigens or
a smear of signal on the membrane. Addition of
protease inhibitors such as phenylmethylsulfonyl
fluoride (PMSF), pepstatin or leupeptin should be
considered.
High background staining
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
46
Poor detection of small
proteins
Small proteins are masked by
large blocking molecules such
as BSA
Consider casein or a low molecular weight polyvinyl
pyrrolidine for a blocking agent
Avoid excessive incubation times with antibody and
wash solution.
Safety Tips:
1. The wires connecting the cell to the power supply must be in good condition, not worn or cracked.
Broken or worn wires not only cause rapid changes in resistance that adversely affects electrophoresis,
but they also create an electrocution hazard.
2. Make sure the area around the power supply is dry.
3. The area for at least 6 inches around the power supply and cell should be bare of clutter and other
equipment. Clear space means any fire or accident can be more easily controlled.
4. Wear gloves while loading and handling the gels; the unpolymerized acrylamide is a neurotoxin.
5. Coomassie blue will stain clothing and hands. In addition it is very acidic. Wear gloves when
handling the staining and destaining solutions.
6. Lab coats or aprons are recommended.
Protocol:
For this experiment, we will follow the protocol for the BioRad Biotechnology Explorer “Comparative Proteomics Kit II:
Western Blot Module, in which students working in pairs will extract proteins from fish muscle tissues, prepare and
load samples in SDS-PAGE, electrophoretically transfer proteins from the gel to a membrane, and locate myosin
proteins by immunodetection. Student pairs will each bring a raw fish tissue from the grocery store to explore the
differences in results from different species of fish, compared to actin and myosin standard proteins. The BioRad kit
instructions will be followed except for the following change: a BioRad Criterion gel electrophoresis apparatus and a
Criterion blotter apparatus will substitute for the smaller Mini-PROTEAN 3 electrophoresis and blotting apparatus
described in the kit manual. Information about the use of the Criterion apparati is available by searching the BioRad
site: www.bio-rad.com, or at the following URLs:
“Criterion Cell Instruction Manual”, Cat # 165-6001 http://www.bio-rad.com/LifeScience/pdf/Bulletin_4006183A.pdf
“Criterion Blotter Instruction Manual” Cat #170-4070
http://www.bio-rad.com/cmc_upload/Literature/34300/4006190b.pdf
DAY ONE:
Lesson 1: Protein Extraction from Muscle
Lesson 2: Electrophoresis – Gel Loading and Running
Lesson 3: Western Blotting – Transfer of Gel Protein Bands to Membrane
There is no good stopping point after loading an electrophoresis gel until the gel has been run and blotted to a
membrane. Blotted membranes can be stored in blotting buffer or wash buffer at 4 oC for up to 1 week.
DAY TWO:
Lesson 4: Immunodetection for Myosin Light Chains
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
47
Technique tips:
To load a sample into a polyacrylamide well,
a. Adjust an automatic pipetter to deliver the correct amount of the sample
b. Attach an ultrathin gel loading tip.
c. Withdraw the correct amount of your sample from your microcentrifuge tube and insert the
pipetter tip into the top of the well to at least four mm of the bottom of the well. Take care not
to puncture the sides or the bottom of the well with your pipetter tip. This takes a steady
hand – it may help to support the micropipettor with your other hand and to support your
elbows on the lab bench top. Make sure that the pipette tip is between the glass sandwich of
the gel and very slowly and gently expel the solution from the pipetter tip into the well while
holding the pipetter steady. The blue solution should fall to the bottom of the well, gradually
filling it.
d. Do not press the pipetter to the second stop – it is important to avoid blowing air bubbles into
the well.
e. Do not release your thumb until you have slowly withdrawn the pipetter tip from the
well, so that you avoid removing the sample that you have so carefully loaded!
Troubleshooting:
 If the sample overflows into the adjacent well, you may be trying to load too much sample.
Alternatively, you may be expelling the sample with too much force, or not withdrawing the
micropipettor tip enough to make room for your sample as it fills the well. You may quickly
withdraw any sample that has overflowed into an empty well.
 Work quickly to minimize the diffusion of samples from the wells or between wells.
Clean up
Be careful not to lose any of the gel apparatus, including the plates, the template, the spacers, and the comb. Wash
them in soapy water, rinse with tap water and dH2O and leave on absorbent toweling to dry.
Dispose of your excess solutions as instructed by instructor.
Remove label tape and any marks made with a marking pen from all glassware. Wash and rinse all used glassware,
give it a final rinse with dH2O, and leave it inverted at your work area in order to drain.
All disposable glassware goes into the special glass disposal receptacle.
REFERENCES:
Andrews, A.T. Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. Oxford Science
Publications, Monographs on Physical Biochemistry. (1986)
Switzer, R. & L. Garrity. Experimental Biochemistry (3rd Ed). W.H. Freeman and Company (1999)
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008
48
Analysis and Questions for Unit 3:
Consult the lab manual Introduction (Unit 1) for a list of topics that your lab report discussion should include.
Your lab report should also include answers to the Focus Questions found in your BioRad Student Lab Manual on
pages 44, 50, 58, and 65. Answers to the following questions should also be added to the end of your lab report:
Lesson 1: Protein Extraction from Muscle
1.
What were the fish species that were extracted by you and your classmates?
2.
Were there any differences in the visible changes in tissues during the extraction procedure?
Lesson 2: Electrophoresis – Gel Loading and Running
3.
What visual clues do you have that a gel box has been assembled correctly and is running as expected?
4.
What visual clues do you have when you are running a gel too hot?
5.
How do you know when to end an electrophoresis run?
6.
How long did your gel electrophorese?
a. How can you speed up the electrophoresis?
b. What limits the speed that a gel can be run?
Lesson 3: Western Blotting – Transfer of Gel Protein Bands to Membrane
7.
Describe the results you would expect if the following problems cropped up during a blotting step and how you
would determine that it had indeed happened:
a. The electroblotting was run too hot.
b. The electroblotting was run too long.
c. The electroblotting buffer was not diluted sufficiently from the stock solution.
d. The electroblotting “sandwich” was facing the anode in the wrong direction.
Lesson 4: Immunodetection for Myosin Light Chains
8.
What differences, if any did you find in the fish proteins when comparing the different species studied by you and
your classmates?
9.
Describe the results you would expect if the following problems cropped up during a blotting step and how you
would determine that it had indeed happened:
a. The blotting solution lacked Tween 20 or milk proteins.
b. The membrane was placed in the staining tray with the blotted side of the membrane on the bottom.
c. The antibodies were prepared or stored improperly and had lost their ability to bind to antigen.
MLAB2479. Molecular Diagnostics Techniques
Unit 3. Analysis of Proteins by Western Blots
ACC Lab Manual, 1st Edition
2008