BIOLOGY 313: CELL BIOLOGY
SPRING 2007
Lab 8: SDS Gel Electrophoretic Analysis of Proteins
INTRODUCTION
In this lab you will use SDS PAGE
(polyacrylamide gel electrophoresis) to
analyze the proteins in Dictyostelium
discoidium cells. SDS PAGE separates
proteins on the basis of their molecular
weights, and there are a variety of proteins
in each of these types of cells. If we
arbitrarily assume that there are ten
different housekeeping proteins found in
both wild type and mutant cells, and there
are four proteins unique to wild type and
six unique to the mutatnt cells, how many
bands are expected in SDS gel patterns of
each of type of cell?
On line simulation SDS gel electrophoresis
http://www.rit.edu/~pac8612/electro/E_Sim.htm
OBJECTIVES
1.
Understand the theory of SDS
PAGE.
You can be certain that you understand the
theoretical basis of SDS gel
electrophoresis if you can answer the
following questions.
Why is there a separating gel and a
stacking gel? What is the function of
SDS? of the -mercaptoethanol? of boiling
the samples? What will be the relative
position on the gel of large and small
proteins? How will migration on the gel be
affected by the % acrylamide? What
S2007BI313Lab8.doc
1
corrections need to be made if the gel
pattern displays “smearing”, “smiles”, no
proteins, all the proteins at the dye front?
Is it possible to recover active proteins
from an SDS PAGE gel? How or why not?
2.
Use SDS PAGE to determine the
molecular weight of the proteins in a
sample.
Your analysis of this experiment is
complete after you have used the
instructions on page 4, Section C: Analysis.
MATERIALS
sample from previous lab
boiling water bath with foam test tube
rack
P10 micropipette & tips
-mercaptoethanol
3x sample buffer
microcentrifuge
1 L DDH O
2
PAGE apparatus with 1 separating gel &
1 sample comb/team, 1 gel chamber/2
teams & 1 power supply/6 teams
Reagents to prepare stacking gel
100 ml 5x running gel buffer
500 ml graduate
“hooked” pastuer pipette
P20 micropipette & tips
1 microwaveable dish & top/ 2 teams
microwave
100 ml Fairbanks A staining solution
100 ml Fairbanks D staining solution
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BIOLOGY 313: CELL BIOLOGY
SPRING 2007
Lab 8: SDS Gel Electrophoretic Analysis of Proteins
PROCEDURES:
A. SDS-polyacrylamide gel sample preparation
1.
Calculate the volume of SDS sample
buffer you will need if you are going to add
one volume of lysed cells to 4 volumes of
sample buffer. Add 5 ul of mercaptoethanol to every 100 ul of SDS
sample buffer that you will need and combine
the cell prep and sample buffer.
2.
Loosen the caps slightly so that they
do not pop off when the tubes are heated.
Load your micro tubes into a Styrofoam rack
and place the rack in a boiling water bath so
that the level of liquid in each tube is below
the surface of the water. Boil the samples
2
for five minutes.(The heat, in combination
with the strong anionic detergent SDS and
the reducing agent 2-mercaptoethanol,
denatures the membrane proteins, unraveling
their tertiary and secondary structures. In
addition, the negatively charged SDS
molecules "coat" protein surfaces, giving them
a strongly negative net charge. As a result,
the proteins will migrate through the pores in
the polyacrylamide gel, from cathode to
anode, primarily according to their molecular
weights.)
3.
Retrieve your samples from the boiling
water bath and tighten the caps. Allow them
to cool to room temperature before loading
them onto the gel.
B. SDS-polyacrylamide gel preparation, electrophoresis and staining.
CAUTION:
Acrylamide and bis-acrylamide are both
neurotoxins and should be handled with extreme care! Always
wear gloves when working with acrylamide or any solution
containing it! Be sure that all acrylamide waste is placed in the
proper receptacles.
1.
Wash the glass plates and spacers
with warm detergent solution, rinse well
with distilled water, and give a final rinse
with acetone.
2.
Assemble the gel apparatus as
instructed by the manufacturer.
3.
At least four hours before the gel is
to be run, mix the separating gel solution
according to Table 1, selecting a
polyacrylamide concentration consistent
with the molecular weight range of the
proteins you are concerned with separating.
Combine all ingredients in the order given
in the table, swirling the flask after each
addition. Add the TEMED and mix gently.
Immediately pour the separating gel to
approximate total height of the gel comb
below the top of the shorter of the glass
plates.
Table 1. Preparation of SDS-polyacrylamide separating gel
(all volumes in ml needed to prepare 2 BioRad miniProteanII gels)
Molecular Weight Range
Deionized water
1.5 M Tris-HCl, pH = 8.8
Acrylamide stock
10% SDS
10% ammonium persulfate (freshly prepared)
TEMED
S2007BI313Lab8.doc
12%
10-100,000
3.35
2.50
4.00
0.10
0.050
0.005
10%
20-175,000
4.10
2.50
3.25
0.10
0.050
0.005
7.5%
40-250,000
4.85
2.50
2.50
0.10
0.050
0.005
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SPRING 2007
Lab 8: SDS Gel Electrophoretic Analysis of Proteins
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.
4.
Within two hours of running the gel,
and after the separating gel has
polymerized, mix 10 ml of the stacking gel
solution according to Table 1. Combine all
ingredients in the order given in the table,
swirling the flask after each addition. Add
the TEMED and mix gently. Immediately
pour the stacking gel to about 2 mm from
the top of the glass plates.
Table 2. Preparation of 4% SDS-polyacrylamide stacking gel
(all volumes in ml needed to prepare 2 BioRad miniProteanII gels)
Deionized water
3.05
0.5M Tris-HCl, pH 6.8
1.25
10% SDS
0.05
Acrylamide stock
0.67
10% ammonium persulfate (freshly prepared) 0.025
TEMED
0.005
5.
Immediately insert the comb into
the stacking gel mixture being careful not
to trap any air bubbles under the teeth of
the comb. Oxygen will inhibit the
polymerization and distort the bottom of
the sample well. Be sure the comb is
centered. Allow the gel to polymerize for
at least 15 minutes.
6.
After polymerization is complete,
carefully remove the comb from the
stacking gel. Pull the comb up with a very
slight side-to-side motion. Be careful not to
disturb the well dividers. Fill each well with
diluted running buffer.
7.
Dilute 70 ml of 5X running buffer
with 280 ml of deionized water for one
electrophoresis run. Add running buffer to
the upper chamber and the lower buffer
chamber: Fill the lower chamber to slightly
above the bottom of the running gel. Use
the “hooked” Pasture pipette to remove any
bubbles from beneath the bottom of the
gel. The gel is now ready to be loaded with
samples.
8.
Load 10 ul of sample prepared as in
Procedure Section B. into each well, using a
predetermined and accurately recorded
distribution of samples into the wells of
the gel(s). Use a micropipettor with
disposable tips, changing tips whenever
changing from one sample to another.
S2007BI313Lab8.doc
Alternatively, use a Hamilton syringe to
load the samples, rinsing the Hamilton 8-10
times with distilled water after each use.
9.
Electrophorese samples at 200v,
constant voltage setting. No voltage
adjustment is necessary for the thickness
of the spacers or the number of gels. The
usual run time is about 45 minutes.
10.
When the tracking dye has reached
the bottom of the gel, turn off the power
supply and disconnect the power cables.
Remove the lid and remove the gels.
l1.
Disassemble the sandwiches (wear
gloves) by gently prying the glass plates
apart by twisting one of the spacers.
12.
The gel will stick to one of the
plates. Leave it there for now. Cut the
stacking gel off with one of the spacers
and discard the stacking gel. Notch the
upper right hand corner of the gel (over
the last well) so that you will know the
proper orientation of the gel once it is
removed. This is very important!!!
13.
Use a Pasture pipette to squirt
Fairbanks Staining Solution A between the
gel and the glass plate; the gel will
gradually slide off the plate. Let it gently
fall into a microwaveable plastic dish with
100 ml of stain for every pair of gels
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SPRING 2007
Lab 8: SDS Gel Electrophoretic Analysis of Proteins
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Cover the plastic dish loosely and
microwave the gels in Fairbanks solution A
16.
.Pour off the second DDH2O rinse,
add 100 ml of Fairbanks Solution D, toss a
crumpled Kimwipe into the dish, cover
for 2 minutes at full power, or to just
short of boiling
loosely and microwave for 1 minute and 30
seconds at full power.
14.
Rock the gels gently for 5 minutes
at room temperature.
17.
Cool for 5 minutes at room
temperature with gentle shaking, discard
the solution and store the gels in DDH2O
until scanning them to produce a permanent
record of the results.
15.
Decant the staining solution into a
container in the hood – the fumes from the
hot isopropyl alcohol and acetic acid are
quite obnoxious – and rinse the gels twice
with 100 ml of DDH2O at room
temperature.
18.
Rinse plates, comb and spacers with
distilled water and place each in the peg
rack when you are done with them.
C. Analysis of SDS-polyacrylamide gels
1.
Lay your stained gel on the CM Rule &
Relative Mobility Calculator (to be handed out
in lab) with the tracking dye on the bottom
line of the Rule (Relative Mobility = 1.0).
Position the gel so the top of the molecular
weight standard lane is aligned with the top
line of the Rule (Relative Mobility = 0). Read
the Relative Mobility of each of the stained
bands in the lane. Record these values and
the molecular weight of each band in the
Molecular Weight standards in the columns of
the Data Sheet that correspond to the lane
number in which the molecular weight
standards were loaded.
2.
Using the semilog paper (to be handed
out in lab) you’ve been supplied, plot the
relative mobility as a function of the log of
the molecular weight for the proteins in the
molecular weight standard sample. (Since the
log scale is on the Y-axis, this graph does not
conform to the convention of plotting the
independent variable on the X-axis and the
dependent variable on the Y-axis. For this
graph, the dependent variable will be plotted
on the X-axis and the independent variable
will be plotted on the Y-axis.)
S2007BI313Lab8.doc
3.
Now determine the Relative Mobility
of each band in the gel lanes that contain your
RBC and WBC samples and record this values
in the appropriate columns of the Data Sheet.
Using the standard curve constructed in steps
1 and 2, determine the molecular weights for
each of these bands and enter the values in
the Data Sheet.
4,
the information from your gels by
answering the following questions.
How many bands are visible in RBC and WBC
samples?
What are the molecular weights of bands
found in both the RBC and the WBC lanes?
What are the molecular weights of the
heaviest bands in each sample? Of the minor
bands?
What are the molecular weights of bands
unique to the RBC lane? To the WBC lane?
Is it possible to provisionally assign identities
to any of the bands in either sample?
Are these assignments consistent with the
major proteins that RBCs and WBCs should
have to accomplish their unique functions?
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BIOLOGY 313: CELL BIOLOGY
SPRING 2007
Lab 8: SDS Gel Electrophoretic Analysis of Proteins
5
REAGENTS AND BUFFERS
Sample Buffer, SDS Reducing Buffer: Store at RT.
deionized water
3.8 ml
0.5 M Tris-HCl, pH = 6.8
1.0 ml
Glycerol
0.8 ml
10 % sodium dodecyl sulfate (SDS) w/v
1.6 ml
2-mercaptoethanol
0.4 ml
1% (w/v) bromophenol Blue
0.4 ml
o
Dilute the sample at least 1:4 with sample buffer, and heat to 95 C for 4 minutes.
1.5 M Tris-HCl, pH = 8.8:
Tris Base/deionized water
27.23 g/~80 ml
o
Adjust pH to 8.8 with 6N Hcl. Make to 150 ml with deionized water. Store at 4 C
0.5 M Tris-HCl, pH = 6.8:
Tris Base/deionized water
6.0 g/~60 ml
o
Adjust pH to 6.8 with 6N Hcl. Make to 100 ml with deionized water. Store at 4 C
5X Running Buffer, pH = 8.3 (dilute 70 ml with 280 ml to make lX):
Tris base
9.0 g
glycine
43.2 g
sodium dodecyl sulfate (SDS)
3.0 %
deionized water
make to: 600 ml
Store at 4oC. Warm to RT before use if precipitation occurs.
Fairbanks staining solution A
Coomassie Blue
Isopropyl alcohol
Acetic Acid
DDH O
2
Fairbanks staining solution D
Acetic Acid
DDH O
2
S2007BI313Lab8.doc
250 mg/250 ml DDH2O
125 ml
50 ml
make to: 500 ml
50 ml
make to: 500 ml
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BIOLOGY 313: CELL BIOLOGY
SPRING 2007
Lab 8: SDS Gel Electrophoretic Analysis of Proteins
DATA SHEET
Team Members:
Lane Sample
1
Dist Rf M.Wt.
Lane
2
Dist
Lane
6
Dist
Lane
7
Dist
Sample
Rf
M.Wt.
S2007BI313Lab8.doc
Sample
Rf
M.Wt.
Sample
Rf
M.Wt.
Lane
3
Dist
Lane
8
Dist
Sample
Rf
M.Wt.
Sample
Rf
M.Wt.
6
Date:
Lane Sample
4
Dist Rf M.Wt.
% Gel:
Lane Sample
5
Dist Rf M.Wt.
Lane
9
Dist
Lane
10
Dist
Sample
Rf
M.Wt.
Sample
Rf
M.Wt.
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Lab 8: SDS Gel Electrophoretic Analysis of Proteins
S2007BI313Lab8.doc
SPRING 2007
7
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