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POLYACRYLAMIDE GEL ELECTROPHORESIS OF BOVINE SERUM
SDS-PAGE, the acronym for sodium dodecylsulfate polyacrylamide gel
electrophoresis, is a popular method for determining the molecular weight of protein
polypeptide chains. SDS, an anionic detergent, will denature proteins by destroying
hydrophobic interactions necessary for maintaining conformation. The SDS will bind with the
polypeptide chain and form a SDS-random polypeptide coil. The ratio of SDS to polypeptide
is believed to be 1.4 grams of SDS per gram of polypeptide and independent of the length
of polypeptide. Quaternary proteins treated with SDS are "broken up" into individual
polypeptide chains, each bound by SDS. SDS-bound polypeptide chains have virtually the
same overall negative charge per molecular size (m.w.) and will migrate in electrophoretic
gels at a rate inversely proportional to the log molecular weight. Methods for determining
molecular weights of polypeptide chains using SDS-PAGE have been perfected by Weber
and Osborn (1969) and Laemmli (1970), the former using continuous gels and the latter,
discontinuous gel conditions. As for most discontinuous PAGE methods, the Laemmli
procedure will result in greater resolution of a polypeptide mixture.
To determine the molecular weight of an unknown polypeptide, a series of molecular
weight protein standards are electrophoresed along with the unknown (in a different lane).
The standard proteins, treated with SDS, of known molecular weights are necessary for
establishing a standard curve of the log molecular weight versus the relative mobility of the
proteins. (See Figure 1) The relative mobility, Rf, is determined by dividing the migration
distance of the polypeptide band by the total migration distance of the running front, which is
made visible by the presence of bromophenol blue. The dye is not appreciably retarded by
the gel pores. A graph of log molecular weights of protein standards versus Rf is linear for a
restricted range of molecular weights for given gel concentrations.
Hames ( 1981) has proposed the following as a guide:
M. W. of Polypeptide
12,000-45,000 daltons
15,000-70,000 daltons
25,000-200,000 daltons
% Acrylamide
15
10
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It is important to stress that the molecular weight determined by SDS-PAGE is the
molecular weight of the polypeptide chain. For proteins that consist of only one polypeptide
chain, it is the molecular weight of the protein; however, for a protein that consists of more
than one chain, more than one band in the gel may result after denaturation.
The various polyacrylamide gels have proven to be the most versatile and useful in
electrophoretic separations because they readily resolve a wide variety of proteins and
nucleic acids. Polyacrylamide gels are formed as a result of polymerization of acrylamide
with the cross- linker N,N' -methylene-bis-acrylamide. The acrylamide monomer and crosslinker are stable by themselves or mixed together, but they polymerize in the presence of an
added free radical system. First, a combination of buffer, monomer, cross-linker, and, in
certain instances, protein is added to the electrophoresis vessel (e.g. tube, slab bed, etc.)
and then polymerization is caused by adding or generating a free radical system in the
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electrophoresis vessel. Biochemists use either chemical or photochemical free-radical
sources to generate gel polymerization. In the chemical method, a free-radical initiator,
ammonium persulfate (APS), is added along with catalyst, N,N,N',N'-tetramethylethylenediamine (TEMED), which catalyzes free radical propagation to the polymerization system.
In the photochemical method, ammonium persulfate is replaced by a photosensitive
compound (e.g. riboflavin) that will generate free radicals when irradiated with UV light.
Current literature contains many electrophoresis procedures and applications for
polyacrylamide gels. Most of them employ polyacrylamide gels in some form of disc gel
electrophoresis or "SDS" gel electrophoresis.
By varying the proportions of the acrylamide and cross-linker, a variety of pore sizes
can be created in the resulting gel. These pores create a molecular sieving effect. The
migration of proteins or other molecules in electrophoresis is due to the presence of
charged ions in the molecules. In proteins, the charges are due to the amino acids radical
groups since the carboxyl and amino groups form the peptide bonds. The types of charges
also depend upon pH so that at pH 6, the basic amino acids (aspartic acid, glutamic acid)
are negatively charged and the acidic amino acids (lysine, arginine, histidine) are positively
charged. Due to the polarity of the electrophoresis chamber, molecules move from the
negative pole to the positive pole based on their net negative charge and/or molecular
weight.
DISCONTINUOUS ("DISC")-GEL ELECTROPHORESIS.
The principles of disc-gel electrophoresis are best illustrated by description of the
step-by-step progress of protein in a disc-electrophoresis system. Consider the "standard"
disc-gel system of Ornstein (1964) and Davis (1964). As shown in Fig. 1 a, disc-gel systems
employ two or more buffer and polyacrylamide-gel conditions in a single gel tube or slab
(stacking gel and separating or resolving gel). Dilute samples of proteins are introduced into
the system from an upper sample layer containing proteins in a stabilizing low-concentration
gel or dense sucrose solution. Voltage is then applied to the initial system after connection
of the gel system between two electrode pools of glycine buffer, pH 8.3. Applied voltage
requires current flow in the form of ion flow in the gel system.
The CI- ions (from the TRIS-HCL) are more negatively charged than the polypeptides
and bromophenol blue which in turn are more negatively charged than the glycine (from the
buffer) molecules. As the CI- ions move through the stacking gel, they leave behind them a
region of low ion concentration. Lowering the ion concentration in the presence of a
constant current increases the voltage gradient. In the presence of a higher voltage
gradient, the polypeptides, bromophenol blue and the glycine ions lag behind as the CIions begin to move faster (driven by the current and their small size).
Therefore, the ions move through the stacking gel in the following order: CI-, then
polypeptides and bromophenol blue, then glycine. The CI- ions create a region of lowvoltage gradient ahead of the polypeptides and bromophenol blue. Any polypeptides moving
ahead into the CI- zone will immediately be slowed down due to the lower-voltage gradient
and thus be overtaken by the larger polypeptide band coming up from behind (Fig 1 b).
Behind the polypeptide band (comprised of the polypeptide species in the sample) is a
region of higher voltage gradient created by the glycine molecules. Any bromophenol or
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polypeptide molecules that move more slowly than the polypeptide band will find
themselves in a region of relatively high-voltage gradient (caused by the glycine) and will
thus move faster and rejoin the polypeptide band.
Initially, glycine anions carrying an average charge of about -0.1 at pH 8.3 move into
the sample and stacking gel region. There the glycine anions encounter the lower pH trischloride buffer and are titrated to a lower pH, pH 6.9. This titration reduces the average (-)
charge on the glycine anions and results in a greatly reduced mobility for the glycine anion
population. In contrast, most proteins have a mobility greater than the glycine ions at pH
6.9, and accumulate ahead of the glycine front. The polypeptide and bromophenol blue
molecules are thereby "stacked" between the leading CI- ions and the trailing glycine ions.
This creates a very fine band of polypeptides and bromophenol blue molecules in the
stacking gel (Fig 1 c).
When all of the ions cross over into the resolving gel with pH 8.9 buffer, migration
events change somewhat. To begin with, the pore size in the resolving gel is a little smaller
than that of the stacking gel. Also, the advancing glycine is titrated back to pH 8.9 by the
higher-strength buffer of the resolving gel. This results in the glycine acquiring a greater (-)
charge and greater mobility with the result that the glycine passes through the proteins. The
CI-,glycine, and bromophenol blue, being small molecules, will not be retarded by the gel
matrixand will thus move far ahead of the polypeptide molecules -the bromophenol blue
remaining between the glycine and CI- zones marking the front of the glycine zone.
Since the polypeptides are large, compared to the other ions, they will be slowed
down by the friction incurred while migrating through the gel matrix. These proteins, each
conveniently restacked in a discrete band, now begin electrophoretic migration in a pH 8.9glycine environment that contains a high concentration of polyacrylamide to enhance the
frictional retardation of the protein molecules. The effect of this is that the polypeptide band
that formed in the stacking gel begins to break up and the different sized polypeptides will
move, according to their size, at different rates in the resolving gel (Fig 1 d).
SDS-GEL ELECTROPHORESIS.
Sodium dodecyl sulfate (SDS)-gel electrophoresis systems are used to resolve and
characterize the number and size of protein chains or protein subunit chains in a protein
preparation. Initially the protein preparation is treated with an excess of soluble thiol (R-SH,
e.g ß-mercaptoethanol) and SDS. Under these conditions, the excess R-SH disrupts
(reduces) all disulfide (-S-S-) bonds present in proteins whereas the detergent SDS binds to
all regions of proteins and unravels all intramolecular protein associations. This results in
total disruption of proteins and total disruption of associated subunit organization and then
yields SDS-carrying, highly-anionic polypeptide chains. The anionic denatured protein
chains are then resolved electrophoretically in a buffered environment that contains thiol
and SDS plus a high concentration of polyacrylamide gel. As a result, the relative mobility of
each of the anionic, denatured polypeptide chains is a log function of the molecular weight
of the polypeptide chain.
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Electrophoretic Characterization of Bovine Serum Proteins
Gel and Buffer Solutions
GEL
Tris-HCl gel, SDS-PAGE
(with -mercaptoethanol
in the sample buffer)
(4-15% gel)
SAMPLE BUFFER
Deionized water
0.5M Tris-HCl, pH 6.8
Glycerol
10% (w/v) SDS
5X RUNNING BUFFER
3.8 ml
1.0 ml
0.8 ml
1.6 ml
Tris base
3.0 g
Glycine
14.4 g
SDS
1.0 g
to 200 ml with deionized
water
-mercaptoethanol *
0.4 ml
0.5 % bromophenol blue 0.4
Note: running buffer should
ml
be- pH 8.3.
8.0 ml Do not adjust the pH
* For best results, add the -mercaptoethanol to the sample buffer immediately before use.
0.5 M Tris-HCl, pH 6.8: add 0.6 g Tris base to 6 ml of deionized water. Adjust to pH 6.8 with
HCl. Make to 10 ml with deionized water.
10% SDS: dissolve 1 g SDS in water with gentle stirring and bring to 10 ml with water.
0.5 % bromophenol blue: dissolve 0.05 g of bromophenol blue in 10 ml deionized water with
gentle stirring.
Staining solution: 1 g Coomassie blue R-250 in a mixture of 500 ml of water, 400 ml
methanol and 100 ml glacial acetic acid (filter before use with Whatman No. 1 filter paper.)
Destaining solution: 500 ml water, 400 ml methanol and 100 ml of glacial acetic acid.
Preparation of Samples and Standards
1. Add 1 µI of bovine serum preparation to 99 µI of sample buffer (1% dilution) in a
microcentrifuge tube. Cover and poke a hole in the top of the tube with a dissecting needle.
Do the same for a 0.5% dilution (1 µI to 199 µI).
2. Prepare a diluted solution of the protein standards by diluting the standards 1 to 20 in the
sample buffer in a microcentrifuge tube (i.e. 5 µI of the standards solution and 95 µI of
buffer). Cover and poke a hole in the top of the tube with a dissecting needle.
3. Heat the samples and standards solutions in the microcentrifuge tubes for 4 min. at 95°C.
This reduces and denatures the proteins.
4. The experiment will use duplicate wells in the gel (the middle 8 wells) with 10 µI of the
standards sample in wells 2 and 6. Place 10 µI of the 1% sample in wells 3 and 7. Place 10
µI of the 0.5% sample in wells 4, 5, 8 and 9.
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Running the Gels
1. Follow the instructions to place the gels in the electrophoresis chamber.
2. Dilute the running buffer (60 ml buffer and 240 ml of water) and load the running buffer
into the chamber.
3. Load the samples as instructed.
4. Place the cover on the chamber and run the electrophoresis at 200 V for about 30-45
minutes, note movement of stained leading edge of samples. Do not let it run off the edge of
the gel.
5. Disconnect the power and then remove the gels.
Splitting the Gels
1. Using a razor blade, cut along the outer edge of the white strips on the smaller plastic
sheet. Carefully lift the smaller sheet leaving the gel on the larger sheet.
2. Locate the space between lanes 5 and 6 and cut the gel in half using a razor blade.
Notch each gel half in the upper left hand corner (close to wells 2 and 6).
3. Place one-half in a dish containing Towbin buffer and one-half in a dish containing
running buffer. The Towbin buffer half will be used in the Western Blot (Consult the
handout: Protein Identification using Western blotting for the next steps for this half
of the gel); the other half will be stained with Coomassie blue stain, a general stain for
proteins.
Staining the Gel
1. Remove the running buffer from the dish by slowly pouring it into a bottle. Make sure the
gel remains in the dish. Pour in enough Coomassie blue stain to cover the gel. Stain the gel
for 30 minutes.
2. Destain with destaining solution to remove background (usually 2-3 hours). Change the
destaining solution as often as necessary until the background is clear.
3. Store gel in zip lock sandwich bags with a little fixative and glycerol to keep it moist.
Analysis
Determine molecular weights of proteins separated on the gel by comparing their position
against the standards. Also compare relative amounts of each protein by observing the
intensity of staining of the bands. Use a biochemistry text or other references to try to
identify the proteins by their molecular weights. Do not automatically use the names of the
standards because they may not be in the sample.
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