Name__________________________
Biology 211 Intro Molecular and Cell Biology Lab
SDS-Polyacrylamide Gel Electrophoresis
Purpose: To learn the principles of SDS-polyacrylamide gel electrophoresis for
the separation of cellular proteins. To identify the major proteins in extracts of
NIH-3T3 cells.
References:
Clark, J. M. and Switzer, R. L. (1977) Experimental Biochemistry, second
edition.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227, 680-685.
Background:
Electrophoresis of proteins in SDS-polyacrylamide gels is a widely used
procedure for the separation of proteins. From gel electrophoresis results, one
can learn the molecular weight of polypeptides, the number of subunits in a
protein or moniter the purification of proteins. In cell biology, SDSpolyacrylamide gels are also used to track proteins in particular cell
compartments. The use of antibodies (specific protein-binding proteins) in
methods such as a Western blot or immunoprecipitation helps us learn even
more about the details of the biochemical processes that take place in cells.
Proteins can be separated in an electric field on the basis of size if they
are first denatured and made soluble with the detergent, sodium dodecyl sulfate
(SDS) and a disulfide bond reducing compound, such as 2-mercaptoethanol.
SDS binds to polypeptide chains, converting them to rod-like shapes and giving
them an overall negative charge. When subjected to polyacrylamide gel
electrophoresis, the proteins are separated according to size by the molecular
sieving effects of the gel. The technique of SDS-polyacrylamide gel
electrophoresis has been widely used to determine the molecular weights of
unknown proteins by comparing their relative electrophoretic mobility to standard
proteins of known molecular weights.
Polyacrylamide gels are generated by the polymerization of acrylamide
monomer and the crosslinking co-monomer N,N'-methylene-bis-acrylamide
(referred to as bis). Variations in the concentrations of monomer and crosslinker
leads to polyacrylamide gels with different pore sizes. For separating proteins,
usually concentrations of 8-12% polyacrylamide are used with ratios of 19:1 or
29:1 of acrylamide to bis. The polymerization reaction is initiated by a catalyst
(TEMED or N,N,N',N'-tetramethylenediamine) and an initiator, ammonium
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persulfate. Unpolymerized acrylamide is a neurotoxin, so please handle gels
with gloves.
Procedure: Week 1
Practicing using micropipets.
The instructor or TA will demonstrate how to use the micropipets. Use a
P20 micropipet to measure between 1-20 ul. Use a P200 micropipet to measure
between 20 ul and 200 ul. Practice pipeting and transfer water samples before
you prepare protein samples.
Preparing protein samples and running the gel.
You will be comparing 3 different protein samples
Sample 1
Prestained proteins
Sample 2
Bovine serum albumin
Sample 3
NIH-3T3 cell extract
1. Obtain samples of the protein standards and bovine serum albumin. These
already contain load solution with SDS and 2-mercaptoethanol.
2. Prepare a sample of your cell extract containing 1-10 ug protein in up to 20 ul
volume. Add an equal volume of load solution.
Cell extract
concentration
(from last week)
Volume of extract
Volume of load
solution
Amount of
protein in
sample
3. Boil your samples at 95C for 4 min. to denature the proteins.
4. Two groups can share a single gel. Remove the comb from the gel. Insert
the gel sandwich into the electrophoresis unit. Another group should insert their
gel on the other side. Fill the buffer chamber with 1x SDS-PAGE buffer. Use a
pipet to rinse out the sample wells with buffer.
5. Inspect the wells to be sure they are intact. Empty wells can be used to
practice loading. Pipet 10 to 20 ul of load solution into these wells to practice
loading.
6. Load the protein samples into the wells after boiling. The samples contain
glycerol which make them more dense than the buffer so they should settle to the
bottom of the wells.
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Group 1
Group 2
Lane 1: Leave empty
Lane 2: Prestained protein standards
Lane 3: Bovine serum albumin
Lane 4: NIH 3T3 cell extract
Lane 5: Leave empty
Lane 6: Leave empty
Lane 7: Prestained protein standards
Lane 8: Bovine serum albumin
Lane 9: NIH 3T3 cell extract
Lane 10: Leave empty
Note that if your gel has one or two bad wells, but is otherwise intact, you should
try to rearrange the samples so that the bad wells are avoided.
7. The instructor will help you connect the leads to the power supply.
Remember that the SDS will give your proteins an overall negative charge, so
they will migrate to the positive (+) pole. For most apparatus, the positive pole is
labeled with red and a red cable is used to connect the positive pole to the power
supply. The negative pole is usually labeled with black and a black cable is used
to connect to the power supply.
8. The gel is run at 150 volts (constant voltage) for 1 hour or until the
bromophenol blue dye in the samples is at the bottom of the gel.
9. When the gel has finished running, the power source should be turned off and
the leads should be disconnected.
10. The electrode buffer can be discarded and the gel units can be dissembled
(WEAR GLOVES!). The instructor will help you trim off the stacking gel and
transfer the gel to a container for staining with Coomassie Blue stain (0.1%
Coomassie Blue R in 40% methanol, 10% acetic acid) in order to visualize the
protein bands in the cell extract and bovine serum albumin lanes.
11. Return after class the next day or at another prearranged time to pour off the
stain solution and add destaining solution (40% methanol, 10% acetic acid).
12. The instructor will dry the gels between cellophane sheets so you can
analyze them next week in lab.
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Name____________________
Assignment: Questions and Gel Analysis
Due in lab Tuesday Nov. 27, 2001.
1. What is the purpose of each of the following chemicals used in SDS-PAGE?
a. Sodium dodecyl sulfate (SDS)
b. polyacrylamide
c. 2-mercaptoethanol
d. prestained protein standards
2. Measure the distances that each of the proteins traveled from the top of the
gel to the middle of the band and complete the table below.
Prestained standards
distance
kDa
175
83
62
47.5
32.5
25
16.5
6.5
Bovine serum albumin
distance
kDa
66.4
4
Cell extract major bands
distance
kDa
3. On the enclosed semi-log graph paper, plot the distance migrated on the Xaxis. Plot the protein size (log10 kDa) on the Y-axis. The instructor will help you
with coordinates.
4. Complete the table with sizes of the major bands (identify the top 2-5 bands)
from the cell extract.
5. Which band in your cell extract might be the cytoskeletal protein actin (43
kDa)? Actin is usually among the most prevalent proteins found in fibroblast
cells.
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