ELECTROPHORETIC SEPARATION OF PROTEINS
I. SEPARATION OF AMINO ACIDS AND PROTEINS BY CHARGE AND SIZE
A. Separation by Charge
All amino acids contain at least two ionizable groups: the amino and carboxyl groups at their
termini. In addition, the R-groups of several amino acids can be charged. These charged R groups are
responsible for the migration of proteins in an electric field. In acidic solutions, amino groups are
positively charged while carboxyls are not ionized. Therefore, in strong acidic solutions, positively
charged amino acids migrate in an electric field to the negative electrode (cathode). In basic (alkaline)
solutions, the carboxyls are negatively charged while the amino groups are not ionized. Therefore, in
strong alkaline solutions, amino acids are negatively charged and migrate to the positive electrode (anode)
during electrophoresis.
Figure 1. General Structure of Amino Acids in Acid and Alkaline Solutions
NH +
NH
3
R
C
H
Acid Solution
COOH
R
2
C
COO -
H
Alkaline Solution
The pH at which an amino acid bears no net charge and does not migrate in an electric field is called
the isoelectric point. Most neutral amino acids have isoelectric points around pH 6.0. The isoelectric
points of acidic amino acids, such as aspartic acid and glutamic acid, are close to pH 3. Therefore, at pH
6, they carry a negative charge and migrate to the positive electrode during electrophoresis. The
isoelectric points of basic amino acids, such as lysine and arginine, are pH 9.7 and 10.8, respectively.
These amino acids carry a positive charge at pH 6 and migrate to the negative electrode. These
differences in charge permit the electrophoretic separation of acidic, neutral, and basic amino acids at pH
6 as illustrated in Figure 2.
The charged side chains of proteins cause them to migrate like amino acids in an electric field. The
electrochemical character of a protein is dependent primarily on the numerous positively charged
ammonium groups (-NH3+) of lysine and arginine and histidine and the negatively charged carboxyl
groups (-COO-) of aspartic acid and glutamic acid. The isoelectric points of most proteins is in the range
of pH 5 to 7. Electrophoresis of proteins is usually performed at a pH above the isoelectric point of most
proteins. The pH of the electrophoresis buffer used in the exercises which follow is 8.6. Most proteins
are negatively charged at this pH and when applied to sample wells at the negative electrode end of the
gel, they travel towards the positive electrode. The rate of migration of a protein species in an electric
field depends upon its charge density (the ratio of charge to mass); the higher the charge density, the faster
the protein will travel. For example, serum albumin, which has an isoelectric point of 4.8, will carry a
strong negative charge in a buffer of pH 8.6 as compared to gamma globulin, which has an isoelectric
point of 7.2. Therefore, at pH 8.6, albumin will migrate toward the positive electrode at a much faster
Electrophoretic Separation of Proteins
Page 2
rate than gamma globulin. Agarose gels are commonly used in the charge separation of proteins since
they form a sponge-like network which serves as a medium for the buffer but has pores large enough to
allow even the largest proteins to pass unimpeded.
B. Separation by Size
Electrophoretic separation of proteins by molecular size requires several modifications in the basic
procedure described above. First, proteins are covered with the anionic detergent, sodium dodecyl
sulfate (SDS), which masks their native charges with its own negative charges. Proteins which contain
disulfide bonds must also be treated with reducing agents (like ß-mercaptoethanol) to cleave these bonds
so that polypeptide chains dissociate from each other and unfold. Proteins that have been treated with
SDS and a reducing agent assume a rod like structure and carry the same charge density imposed by
anionic SDS. Since such proteins no longer possess their native shape and charge, they are referred to as
denatured proteins.
The final modification involves a reduction in the pore size of the agarose gel so that the gel matrix
serves as a molecular sieve. Thus, electrophoretic separation of denatured proteins sorts them according
to size since it relies on the ability of uniformly charged proteins to fit through the pores of gel matrix.
Consider again the electrophoresis of albumin and gamma globulin. Albumin consists of a single
polypeptide of molecular weight 66,000 daltons while gamma globulin is a multipeptide protein
containing two 53,000 dalton polypeptides and two 23,000 dalton polypeptides. Native gamma globulin
Electrophoretic Separation of Proteins
Page 3
migrates toward the positive electrode slower than albumin at pH 8.6 since it carries a weaker negative
charge. When it is denatured, however, gamma globulin has the same charge density as albumin and its
polypeptide chains dissociate from each other. Thus, electrophoretic migration will only be affected by
the sieving property of the gel matrix and the smaller, 23,000 dalton gamma globulin polypeptide will
move the fastest toward the positive electrode, followed by the 53,000 dalton gamma globulin polypeptide
and then by albumin.
Figure 3. Components of a Horizontal Electrophoresis System for the
Separation of Proteins
POWER SUPPLY
+
(Anode)
+
-
Support Medium
(Agarose Gel)
Side
View
(Cathode)
-
Electrophoresis
Chamber
Buffer
-
+
Top View
Sample
Wells
Polyacrylamide gels are commonly used to produce this sieving effect since small pores sizes are
readily attained within the polyacrylamide gel matrix. However, the preparation of polyacrylamide gels is
laborious and necessitates handling a number of toxic chemicals. Therefore, a special type of small pore
size agarose gel which is comparable to the polyacrylamide gel for separating denatured proteins is used
in Experiment 1 - Molecular Weight Determination.
II. PRACTICAL ASPECTS OF ELECTROPHORESIS
The Horizontal Electrophoresis unit and Power Supply
The Horizontal Electrophoresis unit (Figure 3) is composed of an acrylic cell with central platform,
platinum electrodes, four removable gel casting trays, four sample well forming combs and a safety lid
with power cords. The four gels are made in the casting trays and then placed on the central platform of
the electrophoresis cell. Each gel contains 8 sample wells.
Electrophoresis power supplies contain a push button selection panel or selection dial for voltage
Electrophoretic Separation of Proteins
Page 4
outputs. The units can furnish power to one or two electrophoretic cells. An ammeter permits the current
to be monitored during an electrophoretic run. An indicator light alerts the user to an overload situation.
An overload will not occur if the procedures described below are followed.
The Agarose Gel
Several types of stabilizing supports have been used in the electrophoretic separation of proteins.
These include filter paper cellulose acetate and gels composed of either starch, polyacrylamide, agar or
Electrophoretic Separation of Proteins
Page
5
agarose. The agarose gel is an ideal solid support for the separation of proteins on the basis of charge,
and the polyacrylamide gel is generally used for the separation of proteins on the basis of size. However,
due to the hazards associated with using polyacrylamide gels (p. 3), in Experiment 1 - Molecular Weight
Determination, a high percentage agarose gel (5-6%) which contains small pores will be used to separate
SDS treated proteins on the basis of size.
Agarose is a natural polysaccharide of galactose and 3,6-anhydrogalactose derived from agar, which
in turn is obtained from certain marine red algae. Agarose gels are made by dissolving the dry polymer in
boiling buffer, pouring the gels into casting trays and allowing them to set by cooling at room
temperature. The gel forms a porous sponge-like network which serves to hold the buffer and proteins
during the electrophoretic separation. Agarose gels are frequently run in the "submarine" mode where the
gel is completely immersed in buffer. This feature reduces heat development in the gel which could
otherwise lead to protein band distortion.
Electrophoresis Buffer
The pH of the electrophoresis buffer is important in electrophoretic separation, since it will influence
the net charge on the protein molecules in a sample. The pH of the buffer used in these experiments is
8.6, which is higher than the isoelectric points of most proteins. Therefore, most proteins carry a net
negative charge at pH 8.6 and migrate toward the positive electrode during the electrophoretic separation.
The ionic strength of the buffer solution is also important in electrophoresis. Low ionic strength
buffers permit fast migration rates and low heat development. High ionic strength buffers can promote
the sharpening of protein zones, but may also cause high heat production (due to conduction of higher
electrical current) which can lead to band distortion. The moderate ionic strength buffer used in these
experiments permits optimal resolution of protein bands in minimal time.
Staining
Most proteins are not colored, and therefore it is necessary to visualize them in some way in order to
determine their position in the agarose gel after electrophoresis. The stain Coomassie blue, has already
been incorporated into the protein samples in Experiment 1. Therefore, the protein bands will be visible
after electrophoresis. In Experiment 2, the proteins are transferred to a nitrocellulose membrane and then
stained with Ponceau S Protein Blot Stain which stains the proteins red. The staining solutions also
contain acetic acid which serves to precipitate and immobilize the proteins in the gel or nitrocellulose
membrane and prevent blurring by diffusion. The stain is water-soluble and excessive rinsing may cause
destaining. If this occurs, simply place the membrane back into the staining solution and repeat the
staining process.
The Protein Samples
The protein samples are provided in a buffer containing several important components. The buffer
contains 10-20% glycerol which ensures that the samples will layer smoothly at the bottom of the sample
wells. The sample buffer also contains the tracking dye bromophenol blue which enables the investigator
to follow the progress of an electrophoretic run. (In Experiment 1, do not confuse this dye with the
Coomassie Blue which actually stains the proteins.)
The sample buffer in Experiment 1 also contains a reducing agent, - mercaptoethanol as well as
sodium dodecyl sulfate (SDS). The reducing agent eliminates the disulfide bonds while the SDS masks
the proteins native charges with its own negative charges.
III. PROCEDURES FOR THE PREPARATION AND ELECTROPHORESIS OF
Electrophoretic Separation of Proteins
AGAROSE GELS
Page
6
Pouring the Agarose Gels
1. Place the casting tray on a level surface and place a clean glass slide into the gel support deck.
2. Seal both ends of the casting tray with tape. The tape must be firmly pressed against the edges of the tray
to ensure a tight seal and prevent leakage of melted agarose
Electrophoretic Separation of Proteins
Page
7
3. Dispense 15 ml of the appropriate buffer into a 25 ml glass test tube and add the appropriate amount of
agarose. Gently stir the agarose with a glass rod until it forms a suspension.
Experiment 1 - 15 ml of gel buffer (Tris Borate, pH 8.6) and 0.8 grams SDS protein
electrophoresis grade agarose
Experiment 2 - 15 ml of electrophoresis buffer (Tris-Glycine, pH 8.6) and 0.12 grams of protein
electrophoresis grade agarose
4. Place the test tube into a boiling water bath and allow it to boil, stirring it occasionally, until the agarose
suspension is absolutely clear (6-8 minutes). Remove the test tube from the bath, stir gently and cool at
room temperature for about 2-3 minutes.
5. Pour the melted agarose directly from the test tube onto the casting deck, leaving a small amount of
melted agarose in the test tube to use for sample application (see below). Return the test tube to the hot
(but not boiling) water bath. Gently rock the casting deck back and forth one time to evenly distribute the
melted agarose. Insert the comb into the casting tray slots and push down gently on the top of the comb
until resistance is encountered. The teeth of the comb will come to rest in the melted agarose about 0.2
mm above the surface of the glass plate and forms wells in the gel to hold samples.
6. After the gel has cooled for 15-20 minutes (put in refrigerator for faster cooling), remove the tape strips.
The agarose gels can be used immediately, or the gels can be covered with plastic wrap and left overnight
or longer in the refrigerator before use. If they are to be used immediately, carefully lift the comb straight
up and away from the casting tray.
Sample Application
Samples must be loaded very carefully onto the agarose gel in order to obtain accurate results. The
samples are loaded into the wells using precision micropipetors which can be used to dispense 5, 10, 15 or
20 l volumes. (Note that the samples in Experiment 1 must be boiled prior to loading onto the gel.)
To operate the micropipette:
1. Insert the metal plunger into the end of the glass pipet that is opposite the calibration lines. (The
micropipette can be held in one hand and the plunger assembly operated with the other, or you may wish
Electrophoretic Separation of Proteins
Page 8
to hold the micropipet between your thumb and middle finger and operate the plunger with your index
finger of the same hand.)
Gently push down on the plunger until the plunger handle comes to rest on the pipet.
2. Hold the micropipette in a vertical position and place the filling end into the sample solution. Draw the
sample into the pipet to the 10 l calibration line by lifting up on the handle of the plunger assembly.
Micropipet apparatus
3. Carefully wipe excess liquid from the outer pipet surface. Direct the filling end of the micropipet to the
top of a sample well and eject the sample.
4. Rinse the pipet between samples by drawing up and expelling distilled water three times.
5. After all the samples have been loaded into the wells, transfer the melted agarose from the tube in the hot
water bath to a small test tube. Using a glass rod, quickly but carefully transfer a small drop of the
agarose solution to each sample well to seal it.
Electrophoresis - The Quadracell
1. The four casting trays with gels are transferred to a central platform of the electrophoresis cell and
positioned so that the sample wells are closest to the black (negative) electrode. During electrophoresis,
most proteins will then migrate from the negative (black) towards the positive (red) electrodes.
2. Slowly fill the electrophoresis chamber with electrophoresis buffer until the four gels are covered with a 1
cm layer of buffer. Approximately 2.5 liters of buffer are required.
3. Place the electrophoresis cell lid in position.
4. With the power supply off, connect the cables from the cell to the power supply, red to red (positive) and
black to black (negative).
5. Turn on the power supply and set the voltage for 150 V. The voltage will now remain constant at 150 V
during the run.
6. Experiment 1 - Electrophorese until the bromophenol blue in the sample solution has migrated to
within 1.5 cm of the positive
electrode end of the gel. At 150 V, this takes approximately 1 hour.
Electrophoretic Separation of Proteins
Page 9
Experiment 2 - Electrophorese until the albumin (blue bands) in the sera samples and in lane 2
have migrated to within 1.5 cm of the positive end of the gel. At 150 V, this takes approximately 1
hour.
7. At the termination of the electrophoretic separation, shut off the power supply, disconnect the cables and
remove the gel casting trays containing the gels.
8. The buffer should be emptied from the electrophoresis cell and stored under refrigeration in a separate
container until the next electrophoretic run. The same buffer may be used for at least 3 electrophoretic
separations. However, fresh buffer should be used for the preparation of all agarose gels. The
electrophoresis cell should be rinsed with water and stored in an upright position.
Electrophoretic Separation of Proteins
Page
10
IV. SUGGESTED READING AND REFERENCES
PROTEIN BIOCHEMISTRY
1.
Doolittle, R. F. Proteins Scientific American 253:88-100. 1985.
2.
Alberts, B., Bray, L., Lewis, J., Raff, M., Roberts, K. and Watson, J.D. Molecular Biology of the Cell. New York and London:
Garland, 1983.
3.
Richardson, J. S. The anatomy and taxonomy of protein structure. Adv. Protein Chem. 34:167-339. 1981.
4.
Schulz, G. E. and Schirmer, R. H. Principles of Protein Structure. New York: Springer, 1979.
ELECTROPHORESIS
1.
Andrews, A.T. Electrophoresis New York: Oxford University Press, 1981.
2.
Hames, B. D. and Richwood, D. Gel Electrophoresis of Proteins. Oxford: IRL Press, 1981.
3.
Jeppson, J.O., Laurell, C.B. and Frazen, B. Agarose Gel Electrophoresis Clin Chem 25:629-638, 1979.
4.
Nerenberg, S.T. Electrophoresis Philadelphia, F.A. Davis, 1965.
EXPERIMENT I - Molecular Weight Determination of an Unknown Protein
Analysis of a protein frequently involves determination of its size or molecular weight which may be
determined using a variety of techniques. Gel electrophoresis in the presence of SDS is one of the most
common methods since it is relatively simple to perform and does not require elaborate equipment. When
an SDS denatured protein is separated electrophoretically, its rate of migration toward the positively
charged electrode is proportional to the log10 of its molecular weight. When the molecular weights of
several standard proteins are known, their mobilities can be plotted as a function of molecular weight to
give a linear calibration curve (See Fig 1-1). The molecular weight of an unknown protein can then be
estimated from this calibration curve. In practice, protein standards and unknowns are electrophoresed on
adjacent lanes of the same gel. Following electrophoresis, the relative migration of standards and
unknowns are determined and the molecular weight of unknowns calculated.
In this exercise, you will determine the molecular weights of two unknown proteins by comparing
their electrophoretic migration with the migration of standard proteins. The molecular weights of these
standards are given in Table 1-1. The standard and unknown proteins for this experiment have been prestained to allow you to follow their separation during electrophoresis.
Electrophoretic Separation of Proteins
Page
11
Objective: To follow the electrophoretic separation of prestained standard proteins and two
unknown proteins, and to determine the molecular weight of the unknown proteins.
PROCEDURE
Electrophoresis
1. Prepare the agarose gel as described earlier in this handout.
(The lab instructor should prepare the unknown and standard proteins by tapping the tubes to mix the
contents, and then piercing the top of the tubes with a sharp object (tacks are provided) to allow for
escaping steam during boiling. The tubes are then placed in the heat resistant foam rack provided, which
is placed in a large beaker with sufficient boiling water to immerse but not completely cover the body of
the rack. Boil the proteins for 3 minutes.)
2. Load 10 l of the following solutions into the sample wells.
Sample Well
1
2
3
4
5
6
7
8
Sample
Empty
Standard Proteins (m.w. markers)
Unknown Protein I
Unknown Protein II
Sample buffer
Empty
Empty
Empty
3. Electrophorese until the bromophenol blue in the samples has migrated to within 1 cm of the
positive electrode end of the gel. At 150 V, this should take about 1 hour.
4. Digitize the image of the gel using the video camera and computer provided. You will be using
NIH Image software. Follow the instructions provided carefully.
5. Remove the agarose gel from the electrophoresis unit and measure the distance each standard
protein and each unknown protein has migrated in mm from the point of application (sample
well).
Electrophoretic Separation of Proteins
Page 12
6. While you are waiting during the electrophoresis run, you should prepare your
gel for use in the second part of this experiment (page 15), using the same
preparation procedure used for the present experiment. Do not add the samples
into the wells. Store your gel in a zip-lock plastic bag with some extra buffer
for use in the next laboratory period on Western blots. Place it, identified with
your name, in the refrigerator.
Experiment I - Data and Questions
1. Plot the distance migrated by each standard protein as a function of the log of its molecular weight (see
Figure 1-1). You should use Delta Graph Professional software on a Macinstosh computer to do the plot.
It will pay you to become familiar with this software in preparing your lab reports. Determine the
molecular weight of your unknowns from this calibration curve by showing where on your graph the
unknowns fall and by entering in the spaces provided the molecular weight (in kiloDaltons) of your
unknowns.
Unknown 1 ______________
Unknown 2 _________________
2. Explain why in your experiment all of the proteins went toward the same electrode.
3. What was special about the agarose gel used in this experiment that allowed you to measure molecular
weight by the distance the protein moved.
4. What was the role of sodium dodecyl sulfate in this experiment? Could you have measured molecular
weight accurately without it?
5. Why was mercaptoethanol added? What did it do to the proteins structure?
Electrophoretic Separation of Proteins
Page
13
EXPERIMENT II - The Development of the Immune System and the Western Press-Blot
I. Background Information
Blood is a remarkable tissue containing cellular elements (erythrocytes, leukocytes and platelets)
suspended in a liquid medium called plasma. Whole blood or plasma clots upon standing and if the clot
is removed, the remaining straw colored fluid is called serum. Serum has basically the same composition
as plasma except that it lacks certain proteins (fibrinogen and clotting factors) involved in the clotting
process.
Serum contains a variety of small molecular weight components as well as hundreds of different
serum proteins. The serum proteins are usually divided into albumin and globulin classes. Serum
albumin is the major protein found in serum. In humans, the albumin constitutes about 50% of total
serum proteins. Albumin functions in the transport of many substances in blood and plays an important
role in the control of plasma osmolarity. The serum globulins can be subdivided into three major
fractions. These fractions are the alpha, beta, and gamma globulins. Each globulin fraction contains a
large number of different proteins. For example, the protein transferrin is only one of the proteins found
in the beta-globulin fraction. The antibodies are found in the gamma globulin fraction of serum. For this
reason, the antibodies are also called immunoglobulins (abbreviated as Ig). In mammals, there are 5
major classes of immunoglobulins (IgA, IgD, IgE, IgG and IgM). The major class of immunoglobulins in
blood is IgG and this class constitutes about 10% of total serum proteins.
12
The immune system consists of a diverse set of cells, tissues and organs and about 10 antibody
molecules. The major function of the immune system is to protect the organism from viruses, bacteria,
protozoans and larger parasites. When the organisms enter the vertebrate body, macromolecules on their
surface induce the production of specific antibodies that appear in the serum of the infected animal. The
antibodies, in turn, combine with these foreign macromolecules thereby rendering the invading organisms
inactive and noninfective.
The macromolecules that elicit antibody production are called antigens and are most often
proteinaceous in nature. Although antigens are frequently components of foreign organisms, purified
foreign proteins will serve as antigens in that they will
stimulate the formation of antibodies when injected into a
suitable test animal such as the rabbit. Each antigen
possesses features that are recognized by the antibody and
these features constitute the antigenic determinants or
epitopes. An antigenic determinant recognized by an
antibody molecule may be a unique shape or a sequence of
about 5 to 10 amino acid residues on the protein molecule.
It follows that each protein possess a large number of
potential antigenic determinants and when a foreign protein
is injected into an animal, antibodies to different
determinants on the protein are produced and appear in the
serum. This antibody containing serum is called an
antiserum. Thus, an antiserum generated by immunization
with one purified protein usually contains a large number of
different antibodies which recognize determinants along the
protein.
The structure of the most common type of antibody is best represented as a Y-shaped molecule as
shown in the figure to the left. The molecule is composed of two identical light chains (L) and two
identical heavy chains (H). Each L and H chain contains about 220 and 440 amino acid residues,
Electrophoretic Separation of Proteins
Page 14
respectively. The amino terminal ends of the L and H chains come together to form the antigen binding
sites as shown in Figure I. Two identical antigen binding sites are found on each antibody molecule. The
portions of the L and H chains of a given antibody that form the antigen binding sites contain amino acid
sequences which are different from all other antibodies. These so called hypervariable regions are
responsible for the binding specificity of antibodies.
Fetal and newborn mammals do not make antibodies. Thus, a source of antibodies is needed to
combat infection during early postnatal life. In some mammals including humans, antibodies are
transferred from the mothers blood through the placenta to the fetus. These maternal antibodies create
temporary passive immunity which occurs in humans during the first few months of life. After this time,
the newborn’s immune system begins to produce large amounts of antibodies and this condition persists
into adulthood.
In the cow and related ungulates (horse, sheep, goat, etc.), antibodies are not transferred from the
maternal to the fetal circulation because the large antibody molecules cannot cross the placental barrier in
these animals. Thus, fetal serum in these species is devoid of antibodies. Antibodies appear in the serum
of these animals only after the young have suckled and received the first milk. This milk, called
colostrum, contains large amounts of antibodies which are responsible for the transmission of immunity
from mother to young. Correspondingly, the newborn adsorbs these antibodies from the gastrointestinal
tract which is permeable to large protein molecules (like antibodies) during the first few days after birth.
II. The Western Press-Blot
Antibodies are frequently used to identify specific proteins because they recognize unique
antigenic determinants along a protein molecule. For example, human serum albumin injected into a
rabbit makes the animal produce anti-human albumin antibodies. These antibodies are directed against
determinants of human albumin (shapes and/or amino acid sequence) that are not found on the rabbit
albumin molecule. When the anti-albumin serum is mixed with human albumin, the antibodies in the
serum react strongly with the albumin and an antibody-antigen complex is formed. Similarly, the
antiserum will react strongly with albumin isolated from gorilla, orangutan, and baboon. In contract, the
anti-human antiserum will not react with transferrin or any other protein in serum.
In this experiment, you will be comparing serum proteins from various stages in the development of the
cow. First, you will observe the patterns of electrophoretically separated proteins present in sera from
fetal, newborn, and adult cow. You will then perform an immunological procedure known as Western
Blotting. Through a series of steps (described below), this procedure enables the investigator to visualize
proteins that react with a specific antiserum after the proteins have been separated by electrophoresis.
Electrophoretic Separation of Proteins
Page
15
The first step in Western Blotting is to electrophoretically separate the serum protein samples according to
charge in agarose gels. Next, a specialized membrane made of nitrocellulose is placed on top of the gel.
The proteins are transferred to the membrane by forcing them out of the gel by applying gentle pressure,
forming a replica (blot, Figure 2) of the proteins on the gel. Protein blot stain is then applied to the
membrane to visualize the proteins.
Since the antigen-antibody complexes are not colored, they must be treated in some way in order
to visualize them. Enzymes which catalyze the reaction of soluble, colorless substances to insoluble,
colored products are often coupled to antibodies to permit visualization. In this experiment, the antibody
you will use has been coupled to Horseradish Peroxidase (HRP) which catalyzes the reaction diagramed
in Figure 3. Following incubation of the membrane with the HRP-coupled antibody, the final step in the
Western Blotting procedure is to incubate the blot in a color development solution containing hydrogen
peroxide and 4-chloro-1-napthol. The immobilized HRP then converts the 4-chloro-1-napthol to an
insoluble purple product which is deposited at the site of the antigen bands and allows antigen
visualization.
Procedure
I. Preparation of the 0.8% Agarose Gels
1. To prepare one 0.9% agarose gel, dispense 15 ml of electrophoresis butter (Tris-Glycine, pH 8.6) into
a 25 ml glass test tube and add 0.12 grams of agarose. Four gels will be used in the experiment.
2. Boil the agarose and then pour the gel as described on pages 4 - 5.
Electrophoretic Separation of Proteins
Page
16
II. Electrophoresis
1.
Load 15 µl of the following samples into the sample wells.
Sample Well
1
2
3
4
Cow Transferrin
Cow Serum Albumin
Cow Gamma Globulins
Cow Serum
5
6
7
8
Fetal Calf Serum
Fetal Calf Serum
Newborn Calm Serum
Cow Serum
Sample
Group 1
Group 2
2. During the electrophoretic run, perform Step III-1 (below).
3. Electrophorese until the albumin (blue bands) in the sera samples and in lane 2 has migrated to within
1.5 cm of the positive end of the gel. At 170 volts, this should take about 50 minutes.
III. Preparation of the “Western Press - Blot”
1. During the electrophoretic run, perform the following tasks:
A. Wet one sheet of nitrocellulose by floating it in a container with about 20 ml of distilled water.
After wetting, pour off the water and replace with 5-10 ml of TBS. In a separate container, wet two
sheets of blotting paper with 5 ml of TBS.
Note: Gloves should be worn when handling nitrocellulose to prevent transfer of proteins from
your hands to the membrane. If gloves are not available, use forceps. Touch only the edges of the
membranes with gloves or forceps.
B. Cut 6 paper towels (to about 5 cm x 7 cm) to form a stack that is 1-2 cm high.
C. Clean a small section of your laboratory bench top.
2. After electrophoresis, build a “sandwich” on your clean bench top by sequentially overlying each of
the following materials (Figure 2). Smooth with your gloved index finger after each addition except
after step C to eliminate air bubbles between layers.
A. One sheet of wet blotting paper.
B. Agarose gel (place the gel upside down onto the blotting paper so that the nitrocellulose will be in
contact with the bottom of the gel).
C. One nitrocellulose membrane- After the addition of the nitrocellulose membrane, trim a small
corner of the membrane at the bottom of lane 1. This marking will enable you to identify the
positions of the gel lanes on your blot.
Electrophoretic Separation of Proteins
Page
17
D. One sheet of wet blotting paper.
E. One sheet of dry blotting paper.
F. One sheet of dry blotting paper.
G. One stack (1-2 cm) of dry paper towels.
3. Place a gel casting tray, right side up, on top of the stack and press down firmly on the tray for three
minutes. (Note: you should apply moderate steady pressure during this time. Do not press so hard
that the gel collapses completely). During this time, proteins that exit the gel will be trapped on the
nitrocellulose.
IV. Staining the Nitrocellulose Blot
1.
Disassemble the blotting “sandwich”, remove the nitrocellulose membrane and place it in 20 ml of
protein-blot stain.
2.
After 5 minutes, pour off and discard the stain, rinse the blot 3 times with water and note the red
protein bands.
3.
In the space provided below, draw a diagram of the protein bands on your blot and attach a digitized
image of the gel. By examining the positions of the standard proteins on your blots (lanes 1-3), you
should be able to identify these proteins in the sera samples. Label, as best you can, the gamma
globulins, albumins and transferrins on your diagram for each sera sample.
4.
After completion of your diagram, place the blot in 25 ml of gelatin solution and incubate at 37
degrees C for 10 minutes. Replace the solution with 25 ml of gelatin solution and incubate for an
additional 10 minutes. The gelatin serves to block sites on the nitrocellulose that are not occupied by
protein. The dyes will dissociate from the proteins during this step and the blots should appear
colorless at the end of the incubation.
5.
The blots can be processed immediately as described below or they can be stored in gelatin for a few
days in the refrigerator. If stored in the refrigerator, allow them to warm to 37 degrees C before
continuing.
V. Antibody Reactions
1.
Dilute the antibody to Cow IgG (peroxidase linked) by adding 25µl of antibody to 5 ml of fresh
gelatin solution in a petri dish. Transfer the blot to the dish and gently swirl the dish to ensure that all
surfaces of the blot are exposed to the antibody solution.
2.
Place the lid on the dish, and float the dish in a water bath at 37 degrees C for 20 minutes.
Note: Great care should be taken not to bump the dishes during the incubation.
3.
Transfer the blot to a suitable small container (e.g. a clean gel staining tray) and wash for 3 minutes
each in 50 ml of the following solutions. Manual rocking or shaking of the container should be
performed during these washes. Rinse the container with water after each wash.
Electrophoretic Separation of Proteins
1.
2.
3.
4.
4.
Page
18
TBS+NP40
TBS+NP40
TBS+NP40
TBS
While the blots are washing, the instructor should prepare the color development solution by adding 7
ml of development buffer, 0.5 ml of hydrogen peroxide and 5 ml chloronapthol to 130 ml of water.
VI. Color Development Reaction
1.
Place 30 ml of the color development solution into a small container and transfer the blot to the
solution. Gently rock blot in color development solution until purple bands appear. This should take
about 5-15 minutes. Rinse the blot in water and carefully examine both sides for purple bands.
Record in the space below your results by drawing a picture of your gel and by a attaching a digitized
image of the gel. Note the number and relative migrations of proteins that are detected in each
sample. Blots may be stored protected from heat and light (between 2 sheets of black construction
paper, for example).
Electrophoretic Separation of Proteins
Page
19
Experiment II Questions - Answer in space provided
1. Explain the pattern of protein bands observed on the nitrocellulose membrane after the protein blot stain
was applied.
What do the differences in migration distance say about the characteristics and source of the proteins on
the nitrocellulose membrane?
2. Explain the pattern of protein bands observed on the nitrocellulose membrane after the antibody reaction
was completed.
3. What was the purpose of performing the antibody reactions, and what does it tell us about the proteins?
4. Why did the protein bands turn purple when the color development solution was added to them?
6. What does the degree of color intensity on your blot tell you about the amount of protein present versus
the type of protein present?
Electrophoretic Separation of Proteins
Page
20
7. If the color intensity of the stain on your blot were to relate only to the structure of the protein present,
what would this tell you about the relative similarity or difference between it and other proteins that were
colored on the blot?
8. Do you feel that the results of this experiment reliably demonstrate that antibodies are absent in the
serum of young calves before feeding on colostrum? What parts of this experiment are crucial to
demonstrating that the antibodies are, in fact, absent?
9. How could you use changes in the pH of your electrophoresis buffer to improve the separation of proteins,
based on charge, on an agarose gel with large pores? Be sure to center your answer around the concept of
protein isoelectric point.
* Attach answers to questions & additional diagrams, etc. to back if not enough room.