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Introduction to SDS-Polyacrylamide Gel Electrophoresis

Electrophoresis is defined as the transport of charged particles by an electrical field. When
placed in an electrical field, any ion will migrate towards the electrode of opposite charge.
Because most biological polymers carry a net charge at any pH other than their isoelectric point,
they will migrate at a rate proportional to their charge. The electrophoretic mobility (rate of
migration) of a molecule through an electrical field will depend on the strength of the field, and
on the net charge, size and shape of the molecule. Zonal Electrophoresis, the electrophoresis of
polymers in a support matrix, is used both as a preparative and analytical technique in the study
of nucleic acids and proteins. Because the most commonly used support matrices are agarose and
polyacrylamide gels, the more familiar name for this technique is gel electrophoresis.
The support matrix in gel electrophoresis is a porous medium that acts as a molecular sieve. It
retards the movement of macromolecules while allowing small molecules to migrate freely.
Unlike the sieving process of gel filtration chromatography, in which large molecules are
excluded from the matrix, in gel electrophoresis, all of the migrating molecules enter the gel.
Within the gel, larger macromolecules experience a greater degree of retardation and migrate
more slowly than smaller ones. The extent of retardation depends on how closely the gel pore
size approximates the size of the migrating molecules. Agarose gels, which form large pores, are
used to separate very large macromolecules such as nucleic acids, very large proteins, and
protein complexes. Polyacrylamide, which makes a smaller pore gel, is suitable for the
separation of most proteins, and smaller nucleic acid fragments. Polyacrylamide gels are
produced by the polymerization of acrylamide monomers and the cross-linking of the polymers
by the bifunctional agent, bisacrylamide. The greater the concentration of acrylamide (%T), the
smaller the pore size of a gel. Gels are generally prepared as a fixed percentage of acrylamide
(e.g. 5%, 10%, 12%) or as a gradient (e.g. 4-15%). Provided one has an idea about the type of
proteins present in a sample mixture, it is possible to choose a gel formulation that will give the
best possible separation.
In order to use gel electrophoresis to determine the molecular mass of a protein, the molecular
sieving process must be based on size alone. In order for this to occur, all proteins must have the
same charge density (charge to mass ratio) and the same shape. Treatment of proteins with the
anionic detergent, Sodium dodecyl sulfate (SDS), accomplishes this. SDS is an anionic detergent
that denatures proteins and separates the subunits of most multimeric proteins. It is an
amphoteric substance that binds proteins via its nonpolar, hydrocarbon tail, while coating the
protein with the negative charge of its ionic sulfate head group. Most proteins bind the same
amount of SDS per gram of protein, forming molecules of identical charge density. These SDSprotein complexes assume the same rod-like (hairpin) shape, the length of which varies with the
molecular mass of the protein moiety (Figure 1). Thus, in SDS polyacrylamide gel
electrophoresis (SDS-PAGE), SDS-protein complexes migrate toward the anode (the positive
electrode) with a mobility that is exclusively a function of their molecular masses. Glycoproteins
illustrate the most important exception to this principle. The hydrophilic carbohydrate moiety
hinders the binding of SDS and reduces the charge density of the SDS-complexes. Molecular
masses of glycoproteins determined by SDS-PAGE are generally overestimated.
One caution: proteins must be treated with both SDS and a reducing agent with a reactive thiol
group. The two commonly used reagents are -mercaptoethanol (-MSH) and dithiothreitol
(DTT). Either of these reducing agents breaks disulfide bonds in proteins. If such bonds are left
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intact during the formation of SDS complexes, the polypeptide chains are prevented from
forming the "universal" rod-like structure (Figure 2) and correct molecular masses are not
obtained. In addition, in the absence of a reducing agent, the subunits of proteins containing
intermolecular disulfide bonds will not separate and multimeric structures will be retained.

Figure 1. Disruption of quaternary protein structure by SDS. The SDS binds tightly to the subunits obscuring the
original charge on the protein. In effect, all the SDS-protein complexes that form have the same charge/mass ratio.

Figure 2. The role of MSH (or DTT) in the formation of the "universal" rod-like structure of SDS-protein
complexes.

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SDS gel electrophoresis can be used to determine the subunit molecular mass of a protein.
Because the quaternary structure is disrupted, the subunit molecular mass and not the total
molecular mass of a multimeric protein is determined by this method. A minute amount of a
small, anionic dye, bromophenol blue is added to the protein sample. This dye moves fastest analogous to using Blue Dextran on a gel filtration column. The gels are normally run until the
dye front approaches the bottom of the gel. Gels are then stained with Coomassie Blue in
aqueous alcohol. This solution both stains and "fixes" (prevents the proteins from moving) the
gels. The distance that each protein band and the dye front has migrated is then measured and
expressed relative to the migration distance of bromophenol blue. This value, the relative
mobility (Rf), is analogous to the Ve/Vo in gel filtration. It is between 0 and 1 for each protein.
There is a linear relationship between the relative mobility of a protein on an SDS gel and the log
of its subunit molecular mass. A standard curve can be constructed (log Mr versus Rf) using
proteins with known subunit molecular masses (Figure 3). The subunit molecular mass of an
"unknown" protein can be determined from its Rf by interpolation on the standard curve.
5.5

Log Mr

y = -0.9045x + 5.003
R 2 = 0.9772
5

4.5

4
0

0.5

Table 1 Molecular Mass of Standards

Standard Protein
Relative
Molecular
Mass (Mr)
Phosphorylase b
97,000
BSA
66,200
Ovalbumin
45,000
Carbonic Anhydrase
31,000
Soybean trypsin
21,500
inhibitor
Lysozyme
14,400

Rf
Figure 3. Standard Curve for SDS-PAGE

SDS-PAGE can be used to investigate the subunit composition of a protein. If a single band
appears on the gel and its molecular mass is determined to equal the molecular mass of the native
protein, one may conclude that the protein consists of a single subunit (is monomeric). If the
molecular mass of the native protein is a multiple of the subunit molecular mass, one may
calculate the number of subunits present in this multimeric protein. However, the presence of
only one band on an SDS gel does not prove that a protein consists of identical subunits (is
homologous). The protein may consist of heterologous subunits of identical molecular masses
that differ in amino acid composition. The formation of SDS-complexes will obscure the
differences in net charge on these subunits. In addition, molecular mass differences between
subunits that are too small to be resolved on an SDS gel cannot be distinguished by this method.
Heterologous subunits of a protein that can be resolved, will appear as separate bands on the gel
and the molecular mass of each can be determined. SDS gel electrophoresis can be performed on
nonhomogeneous preparations if it is possible to locate the position of the protein of interest. A
method that is routinely used to accomplish this is to transfer the resolved proteins onto a
membrane (Western blotting) and to locate the protein with antibodies, a specific probe.

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The subunit composition of a multimeric protein can be determined by treating protein samples
with a cross-linking agent before applying them to an SDS gel. The cross-linking agent can react
with amino acid residues of two polypeptides that are in contact, thereby linking them by
covalent bonds. This reaction is performed under mild conditions so that not all of the subunits
of each molecule of the protein become cross-linked. The product of this reaction is a mixture
containing molecules representing each possible degree of cross-linking (i.e. monomers, dimers,
trimers, etc). Samples of the treated and untreated protein are applied to the gel for comparison.
If the protein is composed of homologous subunits, the untreated sample will appear as a single
band on SDS-PAGE and the subunit molecular mass can be estimated. The cross-linked sample
will appear as a series of bands of molecular masses equal to that of multiples of the subunit
molecular mass. This is called a ladder. The molecular mass of the intact multimeric protein can
be estimated from the molecular mass of the band with the lowest Rf. The number of subunits in
the protein equals the molecular mass of the protein divided by the subunit molecular mass
(Figure. 4). If the protein is composed of heterologous subunits, deducing subunit composition
by this method can be more complicated but the same principles apply. In either case it is helpful
to have an estimation of the molecular mass of the intact protein by an independent method, such
as gel filtration or equilibrium centrifugation.

100,000 ((tetramer)
75,000 (trimer)
50,000 (dimer)

25,000

25,000 (monomer)

Figure. 4. SDS-PAGE of a protein with and without cross-linking. A plus sign indicates treatment with the crosslinking reagent before electrophoresis; a minus sign indicates no treatment.

Conclusion: This protein is a homologous tetramer (one band indicates homologous subunits).
Subunit molecular mass = 25,000 daltons
Confirming evidence: Molecular mass estimation from gel filtration = 100,000 daltons

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The two kinds of buffer systems used for SDS-PAGE are continuous and discontinuous systems.
In a continuous buffer system, the same buffer (ion composition and pH) is used in the sample,
the gel, and the electrode reservoir and the percent agarose is uniform throughout the gel. The gel
has a relatively small pore size. The sample is applied to the gel (the resolving gel) in as small a
volume as possible and proteins are separated according to size as they travel through the gel. In
a discontinuous buffer system, the sample buffer, gel buffer, and electrode buffers differ in
composition and the gel is composed of two regions. The upper portion of the gel, the stacking
gel, has a relatively large pore size (low percent agarose) and overlays the resolving gel (higher
percent agarose). The sample can be applied in a larger volume than is possible in a continuous
system because proteins will concentrate or stack while they travel through the stacking gel. This
process is indicated by the concentration of the dye front into a straight, sharp line. Because
proteins enter the resolving gel in a narrow zone before they begin to resolve (destack),
discontinuous systems produce better resolution of proteins and are generally preferred.
We will be working with the discontinuous gel system developed by Laemmli, which is the most
commonly used SDS-PAGE system. The sample and the gel contain Tris-HCl buffer, whereas
the electrode buffer is composed of Tris-Glycine. The gels are also known as Tris-Glycine gels
or Laemmli gels and the sample buffer is known as Laemmli sample buffer.
The protein we will be analyzing has a subunit molecular mass in the 20,000-50,000 range.
Resolving power and molecular weight measurements are most accurate in this range on a 12%
resolving gel with a 4% stacking gel. However, because we are using a standard containing
proteins ranging in molecular mass from 10,000 to 250,000 daltons, we will use a gradient gel
instead of a gel of fixed percentage. In this gel, the concentration of acrylamide ranges from 4%
at the top of the gel to 15% at the bottom. This enables the gel to separate proteins over a wide
molecular mass range and produces a standard curve that is linear across the entire molecular
mass range of the standards. Because the upper portion of the gel contains 4% acrylamide and
acts as the stacking gel, there is no need for a separate stacking gel. These gels can be prepared
in a relatively short time. However, because unpolymerized acrylamide is a neurotoxin, it is not
suitable for use in an undergraduate laboratory. Precast gels are commercially available and we
will be using these.
References
1. Nelson, D.L.; Cox, M.M. Lehninger Principles of Biochemistry, 5th ed.; W. H. Freeman: NY,
2008; pp 8890.
2. Hawcroft, D.M. Electrophoresis: The Basics; Oxford University Press: NY, 1997.
.
3. Shi, Q.; Jackowski, G. One-dimensional polyacrylamide gel electrophoresis. In Gel
Electrophoresis of Proteins: A Practical Approach, 3rd ed.; Hames, B.D., Ed.; Oxford
University Press: NY, 1998.
4. Farrell, S.O.; Ranallo, R.T. Experiments in Biochemistry: A Hands-on Approach; Thomson
Learning, Inc.: Pacific Grove, CA, 2000; pp. 207242.
5. Boyer, R. Modern Experimental Biochemistry, 3rd ed.; Benjamin/Cummings: San Francisco,
2000; pp 111121, 133135.

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6. Laemmli, U.K. Cleavage of Structural Proteins during the Assembly of the Head of
Bacteriophage T4. Nature 1970, 227, 680685.

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Biochemistry
Chemistry 354
Laboratry Exercise 5
SDS-Polyacrylamide Gel Electrophoresis
Purpose
1. To determine the subunit molecular mass of yeast alcohol dehydrogenase by SDS-PAGE
2. To determine the subunit composition of alcohol dehydrogenase
Introduction
As presented in the Introduction to SDS-Polyacrylamide Gel Electrophoresis, the molecular mass
of the individual subunits of a protein can be determined from their relative mobility (Rf) on an
SDS gel. This is done by interpolation on a standard curve (log Mr versus Rf) constructed by
running proteins of known molecular masses on the same gel as the protein being analyzed The
protein that will be analyzed in this experiment is alcohol dehydrogenase (ADH), prepared from
baker's yeast (Saccharomyces cerevisiae). It is an enzyme used by yeast in the reversible
fermentation reaction, which converts acetaldehyde to ethanol. A highly purified preparation of
the enzyme will be used in this experiment. Therefore each band that is seen on the gel will
represent a distinct subunit of the enzyme. By using the subunit molecular mass determined in
this experiment and the total protein molecular mass calculated in Problem Set 5, the subunit
composition of the enzyme will be determined.
Protein-SDS complexes are prepared by adding gel sample buffer (GSB) to a solution of protein
in a microcentrifuge tube and heating the tubes in a boiling water bath for 5 minutes. The
samples are then transferred to ice to be cooled. Protein samples must be diluted at least 2-fold
with gel sample buffer, but a higher dilution does no harm. Dry samples may be dissolved
directly into gel sample buffer. The maximum volume that can be applied to a well on the gel
limits the dilution factor. This volume is 30 L on the Bio-Rad mini gels that we are using.
Approximately 0.11.0 g of protein is needed per band for detection of proteins when gels are
stained with Coomassie blue and completely destained. Application of more than 1.0 g per
band is called overloading and will cause bands to be too broad to accurately determine the Rf.
However if the protein consists of heterologous subunits, multiple bands will be present and
more than 1.0 g of protein will need to be applied in order for there to be 0.11.0 g of protein
per band. More protein may also be needed to see bands if the gel is not completely destained.
The composition of the Bio-Rad Precision Plus Protein Standards is shown in Figure 1. The
molecular masses of these proteins have been confirmed by sequencing, mass spectrometry, and
migration in a Laemmli SDS-PAGE system. Approximately 100 750 g of each protein
standard was dissolved in 1.0 mL of gel sample buffer and the SDS-complexes were prepared.
This sample can be stored at -20C for one year. Before use, the solution must to be allowed to
reach room temperature and thoroughly mixed. It is then ready to be applied to the gel. Each
group will be provided with a 30-L sample and 10-L aliquots will be applied to each of two
wells on the gel.

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Figure 1. From the Bio-Rad Laboratory Precision Plus Protein Standards instruction manual

Kaleidoscope Prestained standards (Table 1) are prepared by covalently linking dyes to protein
standards. This makes it possible to monitor the separation of proteins during electrophoresis.
The covalent binding of the dyes to the protein is an uncontrolled reaction, and produces lot-tolot variation in their molecular masses as well as heterogeneity within each sample. Therefore,
these standards are not generally used for molecular mass determinations. Prestained standards
are supplied as SDS-complexes. Before use, the sample is heated for 1 min at 40 C to dissolve
any solid material and then kept at room temperature until it is applied to the gel. Each group
will be provided with a 30-L sample and 10-L aliquots will be applied to each of two wells on
the gel.
Table 1. The composition of Bio-Rad Kaleidoscope Prestained Standards
Protein
Color
Typical Average
Molecular Mass (D)
myosin
blue
202,000
magenta
133,000
galactosidase
bovine serum albumin
green
71,000
carbonic anhydrase
violet
41,800
soybean trypsin inhibitor
orange
25,000
lysozyme
red
16,300
aprotinin
blue
7800
Yeast ADH is provided commercially as a lyophilized solid. A concentrated stock solution of the
enzyme has been prepared as a 1.0 mg/mL solution in sodium phosphate buffer and stored at 20C. You will dilute the sample 1/10 in gel sample buffer and prepare an SDS-complex of the
protein. Samples of this diluted solution containing 0.25 g, 0.50 g, 1.0 g, and 2.0 g will be
applied to the gel. This makes it possible to determine the optimal amount of ADH needed to get
sharp, clearly visible bands.
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Procedure
Materials
Mini-PROTEAN 3 Electrophoresis Module
Power supply
Precast gel: 4-15% gradient gel in 0.375 M Tris-HCl, pH 8.8
Running Buffer: 25 mM Tris base 0.192 M glycine 0.1% SDS, pH 8.5
Laemmli Sample Buffer: 62.5 mM Tris-HCl, pH 6.8 containing 2% SDS, 25% glycerol,
0.01% bromophenol blue
Gel Sample Buffer (GSB): Laemmli Sample Buffer containing 50 mM dithiothreitol
Bio-Safe Coomassie Blue Stain
Bio-Rad Precision Plus Protein Standards (unstained)
Bio-Rad Kaleidoscope Prestained Standards
Yeast alcohol dehydrogenase (Sigma) 1.0 mg/mL in 10 mM sodium phosphate buffer, pH 7.5
Hot plate
Water bath:250 mL beaker containing glass beads, covered with aluminum foil
Ice bucket
Microcentrifuge
Microcentrifuge tubes
250-mL graduated cylinder
Wash bottle of distilled water
Forceps
Scalpel
Spatula
Timer
Marking pen
Digital micropipets (P-20, P-200) and tips
Transfer pipet
Waste beaker
Shaker
Staining Tray
Identi-Plugs for 613 mm tubes
Six-inch ruler
Hazards
The running buffer contains Tris, glycine, and SDS, which may cause skin and eye irritation.
SDS is harmful if swallowed. Laemmli sample buffer contains Tris, glycerol, and bromophenol
blue, which may cause skin and eye irritation. Glycerol and bromophenol blue are harmful if
swallowed. The gel sample buffer contains dithiothreitol, which is toxic and harmful if
swallowed. It targets the central nervous system. It is irritating to the eyes, respiratory system
and skin and has an unpleasant odor. Concentrated solutions should be handled in a fume hood.
Bio-Safe Coomassie stain, Kaleidoscope Prestained Protein standards, Precision Plus
Protein standards, and Ready gels have no irritating effects if handled according to
specifications. Safety glasses should be worn at all times. Gloves should be worn when working
with solutions containing DTT and when staining, destaining, and handling the gel

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Methods
Sample Preparation
1. Fill an ice bucket with ice.
2. Pick up a sample of ADH, GSB, and the molecular mass standards and transfer them to your
microcentrifuge tube rack. The standards are ready to apply to the gel. The ADH sample
must be converted to an SDS-protein complex.
3. Pick up a 250-mL beaker containing glass beads and covered with a sheet of aluminum foil.
4. Punch a small hole in the center of the aluminum foil so that a 1-mL microfuge tube can be
suspended from the foil and not fall into the beaker.
5. Add water to a height that will cover the bottom of the microcentrifuge tube when it is
inserted into the hole in the aluminum foil.
6. Place the 250-mL beaker with the glass beads, the water, and the foil cover on the hot plate
and set the heat setting on about three-quarters of the maximum setting.
7. Dilute 10 L of ADH 10-fold in gel sample buffer.
8. Be sure that the microcentrifuge tube containing the ADH sample is shut tightly.
9. When water is boiling, place the microcentrifuge tube containing the ADH into the hole in
the foil so that it is suspended in the boiling water bath.
10. Boil sample for 5 minutes. Remove the microcentrifuge tube from the bath (you may want to
use a forceps to avoid burning your fingers) and place it on ice to cool.
11. When the sample has cooled, transfer it to the microcentrifuge. Be sure that the tube is
balanced against a tube from another group or a blank tube (containing water). Centrifuge for
one second (or momentary setting). This is to bring any water droplets that have condensed
near the top of the tube back down to the bottom of the tube.
Assembly of the Mini-PROTEAN 3 Module:
Follow the instructions adapted from the Bio-Rad instruction manual (pp.124128). Pay
particular attention to the following modifications listed briefly on page 127:
1. After adding running buffer to the upper chamber, allow the clamping frame to sit on paper
towels for a few minutes in order to check for leaks. The paper towel should remain dry.
2. After adding 200 mL of buffer to the tank, tip the electrophoresis module sideways to allow
any bubbles that are adhering to the bottom of the gel plate to rise to the surface. The
presence of large bubbles can insulate the gel from electrical contact.
3. Add an additional 200 mL of buffer to the lower chamber. Buffer contacting the gel plate
will help cool the plate and prevent overheating during the run.
Sample Loading
Refer to the Bio-Rad instructions for the sample loading technique.
Load the samples according to the following protocol:

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Table 2. Gel Loading Protocol for SDS-PAGE
Well
1
2
3
4
Sample
GSB K
ADH (0.10 mg/mL)
10 2.5
5.0
Amt(L) 10
0.25
0.50
Amt (g)

5
P
10

6
7
ADH (0.10 mg/mL)
10
20
1.0
2.0

8
P
10

9
K
10

10
GSB
10

Note: P = Precision Plus Protein Standards, K = Kaleidoscope Prestained Standards, GSB = Gel Sample Buffer

Running the Gel

1. Run at 250300 V on the constant voltage setting. The current should start at approximately
6580 mA/gel and drop to approximately 50 mA/gel at the end of the run.
2. Run until the dye front reaches the reference line at the bottom of the gel. The run should
take approximately 15 minutes. Try not to let the dye front run off the gel, but it is not a
disaster if it does. You will use the lowest molecular mass standard that you can clearly see
as a reference point instead of the dye front because the position of the dye front is not clear
after staining with Bio-Safe stain.
Staining the Gel
1. Add 200 mL of distilled water to the staining tray.
2. Use the opening lever to open the plates. Insert the end of the lever with the arrow between
the plates at the four arrows on the cassette. At each of the four corners of the plate, use the
teeth on the lever to wedge between the plates of the cassette and crack the plates open using
an upward or downward motion. Once all four corners of the plate are cracked, peel the
plates apart. The gel should remain on the larger plate; discard the short, inner plate.
3. Invert the gel over a staining tray, containing 200-mL of distilled water and the gel should
peel from the plate into the water. If necessary, pry a small corner of the bottom edge of the
gel up from the cassette and remove the gel from the cassette by letting it slide gently into the
tray. Touching the gel to the water combined with gravity will help.
4. Label the tray, cover it, and put the tray on the shaker for five minutes.
5. Wash two more times for 5 minutes with 200 mL of distilled water in order to remove the
SDS from the gel.
6. Replace the distilled water with 100 mL of staining solution and shake for 30 minutes.
7. Discard the staining solution, rinse the gel in distilled water and keep in distilled water to
destain.
8. The bands of protein should be clearly visible immediately after removing the stain.
Measurements can be taken immediately, but the gel should be left to destain if time permits.
To destain, float foam plugs above the gel to adsorb the stain and place the tray on the
shaker.
Measurements and Calculations
1. Remove the gel from the distilled water by lifting the basket out of the tray. If bands are
clearly visible, you can take your measurements. If the gel begins to dry while you are
working, it will curl up. Use a wash bottle to wet the gel with distilled water as needed.
2. Decide which lane of the four containing ADH is the best one to use for measurement. Select
the lane containing the sharpest band that is clearly visible. Select the lane containing the
Precision Plus standards that is closest to the lane containing the ADH sample that you have
selected for measurement.

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3. Measure the migration distance for each protein band by measuring the distance from the
bottom of the well to each band of protein. Do this for each standard protein in the lane that
you have chosen and for ADH.
4. Calculate the Rf for each band by dividing its migration distance by the migration distance of
the fixed reference point you have chosen. Although this reference point was traditionally the
dye front, the staining method that we are using changes the appearance of the dye front from
a sharp blue line to a broad streak. It therefore cannot be used as a reference point and you
should use a standard instead. Use the lowest molecular mass standard that is clearly visible.
The program will recognize its migration distance as the maximum migration distance or
"Max". Note that the Rf of your reference band will be 1.00. Be sure you understand how
many significant figures to include in these measurements before you proceed. This value is
limited by the accuracy of your measurements of migration distance, not by the molecular
mass of the standards. The molecular masses of the standards are known to the nearest
dalton. For instance, standard #1, of molecular mass 250 kD is actually known to be 250,000
daltons.
5. Record this data in the graphing software file that you choose (explained below). The table
will look similar to the one shown below.
Table 3. Data for the SDS-PAGE Standard Curve
Standard
Migration
Rf
Distance (mm)
1
2
3
4
5
6
7
8
9
10

Molecular
Mass (kD)
250.000
150.000
100.000
75.000
50.000
37.000
25.000
20.000
15.000
10.000

Log Mr
2.398
2.176
2.000
1.875
1.699
1.568
1.398
1.301
1.176
1.000

ADH
Three Microsoft Excel templates have been created for this experiment. They are located in
the Biochemistry Graph Template folder on the computers in room 315. See page 5 of this
manual for instructions on where to find these files. Depending on how the gel runs, you may
be able to distinguish 8, 9, or 10 standards. Because of a software problem, the program can't
function properly in a situation where one of the variables is derived (Rf) and the number of
data points varies. Therefore, three distinct files have been programmed that contain 8, 9, or
10 standards. Select the file that is appropriate for your data. The molecular mass of each
standard has been entered into the program and the values of log Mr have been calculated.
All you need to do is enter your migration distances and the program will calculate the Rf by
dividing each migration distance by the maximum migration distance (that of the lowest
molecular mass standard).

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6. The Excel program will create a standard curve by plotting log Mr versus Rf for the standard
proteins and draw the best straight line through the data points by using regression analysis.
It will report the equation for the line and its R2 value. A good straight line should have an R2
value of 0.970 - 0.999. This means that the data points fit on the line well. Note that the same
defect in the software described above causes the program to create a default (bogus) graph
before you enter your data. The equation for this line is y = -0.15x + 2.48. Ignore it. The
correct graph and equation will be displayed when your data is entered.
7. Use the standard curve to estimate the subunit molecular mass for ADH. This is done by
solving the regression equation for x = the Rf for ADH and getting the antilog of your y
value. The program will do this for you and the value for the subunit molecular mass of ADH
should appear in the data table as soon as you enter your measurements.
8. Calculate the accepted value for the subunit molecular mass of ADH from the information
available on the ExPASy web site (4). Enter "alcohol dehydrogenase baker's yeast" into the
search box. You will get 15 hits, five of which (ADH1-ADH5) are the isozymes of ADH
from Sacchromyces cerevisiae. Select ADH1, the predominant form in metabolically active
baker's yeast. The subunit molecular mass that is reported here has been calculated from the
amino acid sequence. This is the subunit molecular mass of the unprocessed apoprotein.
Subtract the molecular mass of the N-terminal extension in order to obtain the molecular
mass of the processed apoprotein. Note that the protein is modified in two ways: it is
covalently modified and contains zinc. Add the contribution to the subunit molecular weight
from the covalently bound group and the zinc atoms to obtain the true subunit molecular
mass of ADH. Ignore the phosphoryl groups in your calculation. These are added in response
to a cell signal under specific conditions and are not present on the protein that was used in
this lab.
9. Use the accepted value of the subunit molecular mass to calculate the percent error in your
estimation.
Photographing the Gel (Gel Doc-it System)
A. Set-up
1. Turn the power on and place a flash drive into the USB drive.
2. Place the Coomassie Blue filter in the filter tray.
3. Select the transilluminator as the light source (Trans) and wavelength (365 nm).
4. Roll out the transilluminator from the dark room and place the UV to white light
converter plate on the transilluminator.
5. While wearing gloves, center the gel on the converter plate.
6. Roll the transilluminator back into the dark room.
7. Turn on the UV lamp and shut the door. You can see the gel through the viewport

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B. Photographing the gel
Note: Use the touch-pen to operate software controls. The pen is kept in a holder, attached
to the right side of the instrument.
1. Select LIVE from the touch screen to see the image of the gel.
2. Rotate the aperture (upper ring on the camera) and adjust the exposure time (+ and keys
on the touch screen) to optimize the brightness of the image.
3. Rotate the zoom lens adjustment (middle ring on the camera) so that the gel fills the
frame and the image is as big as possible.
4. Rotate the focus adjustment (lower ring on the camera) so that the image is in clear focus.
Note: While the program is in the LIVE mode (LIVE button is yellow), the image will
automatically update after each adjustment. Wait for the screen to refresh before making
additional changes.
5. When you are ready to take a photograph, change the wavelength selection to 320 nm.
6. Save the image
a. Select TIME STAMP to add a code number and date stamp to the photo.
a. Select SNAP to capture the image.
c. Select SAVE to save the image to the flash drive. The software has been preprogrammed to save to the flash drive as a jpg file. Other options can be selected
from the Preferences menu.
d. When the image has been saved, the SAVE button will revert from yellow to white,
indicating that the flash drive may be removed from the USB port.
7. Insert the flash drive into a computer and drag the file to the Photoshop icon (the feather)
in the dock to open the file in Adobe Photoshop.
8. From the Image menu, select Brightness and Contrast. Decrease the brightness and
increase the contrast of the image.
9. From the Image menu, select Size. Adjust the width of the image to match that of the gel
(3.5 inches).
10. Use the selection tool on the left toolbar to create a tight border around the gel and
exclude the staining tray from the image. From the Image menu, select Crop to complete
this task.
11. Save the edited image on your flash drive and print copies of the photograph for your lab
report.
12.

Discard the gel in the trash and wash the electrophoresis box, clamping frame, electrode
core, and buffer dam. Rinse the equipment in distilled water and leave it to drain on
clean paper towels.

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Report
Your lab report should include the following:
Figures and Tables
1. Table 1. Data for the SDS-PAGE Standard Curve
2. Figure 1. Standard Curve for the Subunit Molecular Mass Determination of ADH
Report
1. Your estimation of the subunit molecular mass of ADH
2. The accepted value of the subunit molecular mass of ADH
3. Your percent error
4. Calculate the total number of subunits in ADH.
a. Hint: There is information in Problem Set 4 that will help you with this calculation.
b. If the error in this experiment is large, the experimental value of the subunit molecular
mass will give erroneous results in this calculation. If this occurs, use the accepted value
of the ADH subunit that you obtained from the ExPASy site.
5 What is the subunit composition of ADH? Use the appropriate term and explain how you
determined this.
6. What is the optimal amount (in g) of ADH to run on this gel?
References
1. Molecular Weight Determination by SDS-PAGE; tech note 3133; Bio-Rad Laboratories:
Hercules, CA, 2004.
2. Using Precision Plus Protein Standards to Determine Molecular Weight; tech note 3144;
Bio-Rad Laboratories: Hercules, CA, 2004.
3. Dayhoff, M.O. Atlas of Protein Sequence and Structure; The National Biomedical Research
Foundation: Silver Spring, MD, 1972; Vol 5, pp D82.
4. ExPASy Protein knowledgebase: http://www.uniprot.org
5. Leskovac, V.; Trivic, S; Pericin, D. The three zinc-containing alcohol dehydrogenases from
baker's yeast, Saccharomyces cerevisiae. FEMS Yeast Research 2002, 2, 481494.

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Instructions for the Set-Up and Assembly of the Mini-Protean 3 Electrophoresis System
General Information
Introduction
The Mini-Protean 3 cell runs both hand cast gels and precast gels (Ready gels or TGX gels)
interchangeably. The Mini-Protean 3 system includes a casting stand and glass plates with
permanently bonded gel spacers that simplify hand casting and eliminate leaking during casting.
The cell can run one or two gels, and the mini tank is compatible with other Bio-Rad electrode
modules for tank blotting, 2-D electrophoresis, and electro-elution.
Components
Buffer Dam
The molded, one-piece buffer dam is used when running only one gel.
Electrode Assembly The Electrode Assembly holds the Gel Cassette Sandwich. It houses
the sealing gasket, the upper and lower electrodes, and the connecting
banana plugs. The anode (lower electrode) banana plug is identified
with a red marker and the cathode (upper electrode) banana plug with
a black marker.
Clamping Frame
The Clamping Frame holds the Electrode Assembly and Gel Cassette
Sandwich in place. Its pressure plates and closure cams seal the Gel
Cassette Sandwich against U-shaped gaskets on the Electrode
Assembly to form the inner buffer chamber.
Inner Chamber
The Electrode Assembly, two Gel Cassette Sandwiches or one gel
cassette sandwich and a buffer dam, and the Clamping Frame form the
Inner Chamber.
Mini Tank and Lid The Mini Tank and Lid combine to fully enclose the inner chamber
during electrophoresis. The lid cannot be removed without disrupting
the electrical circuit. The Mini Tank and Lid are also compatible with
other Bio-Rad electrode modules for blotting, first dimension 2-D, and
electro-elution.
Set up and Basic Operation
A. TGX Gel Cassette Preparation
Note: The Mini-Protean 3 cell is guaranteed for use only with Bio-Rads Ready Gel or TGX
precast gels.
1. Remove the TGX gel from the storage pouch by gasping the pouch firmly and tearing at
one of the notches.
2. Remove the tape at the bottom of the cassette by peeling it off the bottom edge of the
cassette.
3. Remove the comb by pushing it up grasping the comb from the notch at the center and
lift the comb straight up. Rinse the wells thoroughly with either distilled water (for a
practice set-up) or running buffer (for a run).
4. Repeat for a second gel.
Note: If only one gel is to be run, use the mini cell buffer dam to close the inner chamber.

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B. Mini-Protean 3 Electrophoresis Module Assembly
1. Remove the Clamping Frame from the Mini-Tank. Rotate the cams of the Casting Frame
outward to release the Electrode Assembly from the Clamping Frame (Figure 5a).
2. Place a TGX gel cassette into the slots at the bottom of one side of the Electrode
Assembly. Be sure that the short plate of the TGX gel cassette faces inward toward the
notches of the U-shaped gaskets (Figure 5b).
3. Place the plastic buffer dam on the other side of the Electrode Assembly. Position the
buffer dam so that the writing on the plastic faces inward towards the gaskets. If you can
read the writing the buffer dam is facing the wrong way.
4. Press the gel cassette and the buffer dam up against the green gaskets. The cassette,
buffer dam, and the Electrode Assembly fit together to form the inner chamber of the
electrophoresis unit.
5. Slide the Electrode Assembly, with the plates into the Clamping Frame (Figure 5c).
6. Press down on the Electrode Assembly while closing the two cam levers of the Clamping
Frame. This closes the Inner Chamber and ensures a proper seal of the short plate against
the notch on the U-shaped gasket (Figure 5d). The short plate must align with the notch
in the gasket.

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Note: Gently pressing the top of the Electrode Assembly forces the top of the short glass
plate on the gel cassette to seat against the rubber gasket properly. If you do not push
down, the upper buffer may leak, creating a short circuit.
7. Lower the Inner Chamber Assembly and Clamping Frame into the Mini Tank. Fill the
inner chamber with approximately 125 mL of running buffer, so that the buffer reaches a
level between the top of the short plate and the top of the long plate of the gel.
Note: Do not overfill the inner chamber so that the buffer spills over the top of the long
plate. Overfilling may cause siphoning of the buffer, resulting in buffer loss and
interruption of electrophoresis due to a short circuit.
8. Add 200 mL of running buffer to the Mini Tank (lower buffer chamber), perform a leak
test, remove air bubbles from the bottom edge of the gel cassette if necessary, and add an
additional 200 mL of running buffer to the Mini Tank.
C. Sample Loading
Note: The sample wells are outlined and labeled for easy sample loading and sample
identification.
1. Load samples using an L-10 digital micropipette.
2. The Sample Loading Guide assists in aligning the pipette tip and the wells. If using the
Sample Loading Guide, place it between the gel cassette and buffer dam in the Electrode
Assembly. Insert the pipette tip into the slots of the guide and fill the corresponding
wells.
Note: Load samples slowly to allow them to settle evenly on the bottom of the well. Be
careful not to puncture the bottom of the well with the pipette tip.

Figure 6. Using the Sample Loading Guide

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D. Gel Electrophoresis
1. Remove the Sample Loading Guide and place the Lid on the Mini Tank. Make sure to
align the color coded banana plugs and jacks. Matching the jacks on the lid with the
banana plugs on the Electrode Assembly makes the correct orientation. A stop on the lid
prevents incorrect orientation.
2. Insert the electrical leads into a suitable power supply with the proper polarity. Insert the
red plug into the red slot in the power supply and insert the black plug into the black slot.
If this orientation is reversed, the samples will migrate out of the wells into the buffer of
the inner chamber when electrophoresis begins.
3. Apply power to the Mini-Protean 3 cell and begin electrophoresis; 200 volts at constant
voltage is recommended for SDS-PAGE. Run time is approximately 15 minutes for TGX
gels.
E. Gel Removal
1. After electrophoresis is complete, turn off the power supply and disconnect the electrical
leads.
2. Remove the tank lid and carefully lift out the Inner Chamber Assembly. Pour the buffer
from the Inner Chamber out into the tank.
Note: Always pour off the buffer before opening the cams to avoid spilling the buffer.
3. Open the cams of the Clamping Frame. Pull the Electrode Assembly out of the Clamping
Frame and remove the gel cassette.
4. Use the opening level to pry open the plates of the gel cassette. Insert the end of the lever
with the arrow between the plates at the four arrows on the cassette. At each of the four
corners of the plate, use the teeth on the lever to wedge between the plates of the cassette
and crack the plates open using an upward or downward motion. Once all four corners of
the plate are cracked, peel the plates apart. The gel should remain on the larger plate;
discard the short, inner plate.
5. Invert the gel over a staining tray, containing 200-mL of distilled water and the gel
should peel from the plate into the water. If necessary, pry a small corner of the bottom
edge of the gel up from the cassette and remove the gel from the cassette by letting it
slide gently into the tray. Touching the gel to the water combined with gravity will help.
6. Rinse the Mini-Protean 3 cell electrode Assembly, Clamping Frame, Mini Tank, buffer
dam, and gel loading guide in laboratory soap solution. Rinse thoroughly with tap water,
and rinse again in distilled water. Leave all equipment on paper towels to air dry.

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Problem Set 5: SDS PAGE and Subunit Structure of Proteins
1. The enzyme, glutathione peroxidase, elutes from a calibrated Sephadex G-100 column at a
position corresponding to a molecular mass of 88,000. When this enzyme is run on SDS gel
electrophoresis a single band with a molecular mass of 22,000 is observed. What conclusions
can be drawn about the structure of glutathione peroxidase?
2. The subunit composition of a multimeric protein can be deduced using SDS-PAGE in
conjunction with a protein cross-linking agent. This bifunctional reagent can react with
amino acid residues of two polypeptides that are in contact, thereby linking them by covalent
bonds. After limited treatment with the reagent, so that some but not all subunits become
cross-linked to their neighbors, the protein is subjected to SDS-PAGE and the molecular
masses of the resulting bands are estimated. The results of such experiments on two different
proteins are shown in the figure below. What is the most likely subunit structure of each
protein?
Protein A

Protein B
+

85,000
60,000
50,000
40,000
35,000
20,000

Figure 1. SDS-PAGE of two proteins with and without cross-linking. A plus sign indicates treatment with the crosslinking reagent before electrophoresis; a minus sign indicates no treatment

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3. You have a protein that is a heterologous trimer of two subunits of molecular mass, 20,000
and one subunit of molecular mass, 15,000. On the figures below, draw the results you would
expect to obtain if the protein is run on SDS-PAGE before () and after (+) limited treatment
with a crosslinking reagent. Show the protein bands you would expect to see and indicate
their molecular masses.

Barbara T. Nash, Ph.D., Mercy College September 2012