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1
SDS – PAGE GELS (Protein)
I.
Solutions Required for Gel
A.
30% Acrylamide 100 ml (wear gloves and mask).
Acrylamide
29.2g
Bis-Acrylamide
0.8g
dH2O
50 ml (to dissolve)
Cover from light and allow to stir until it dissolves. Bring volume up to 100 ml and filter.
Keep in brown bottle at 4°C (good for 30 days)
B.
2.0M Tris-Cl pH8.8 100 ml
Tris Base
24.22g
dH2O
50 ml (to dissolve)
Dissolve and adjust pH to 8.80 with concentrated HCl. Bring up volume to 100
ml and filter. Store at 4°C.
C.
60% Sucrose
Sucrose
60g
Bring up the volume to 100 ml.
Store at 4°C
D.
10% SDS
SDS
50g
dH2O
400 ml (to dissolve)
Dissolve SDS in water – place in warm water to help it dissolve. Bring up to 500 ml and
store at ROOM TEMPERATURE.
E.
0.5 M Tris-Cl pH 6.8
Tris-Base
6.055g
dH2O
50 ml (to dissolve)
Adjust pH to 6.8 with concentrated HCl. Bring up the volume to 100ml. Filter and store
at 4°C.
F.
SDS Electrophoresis Running Buffer
1 liter
0.33M Tris
4.0g
0.256M Glycine
19.2g
Adjust topH8.8 w/ dil NaOH. Do not add HCl
Add 10% SDS
13.3 ml
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G.
2
Coomassie Destain Solution
40% Methanol
10% Acetic Acid
50% dH2O
H.
Coomassie Stain
0.1% (wt/vol) Coomassie Blue Stain in Coomassie Destain Solution
Stir overnight and filter
I.
Blotting buffer
Concentration
For 1 liter
0.25M Tris(base)
3.03g
0.192M Glycine
14.42g
20% MeOH
200ml
pH should be 8.3 – Check it to make sure it is fine but DO NOT adjust
J.
Tris Buffered Saline (TBS)
Concentration
For 1 liter
20mM Tris (base)
2.43g
500mM NaCl
29.24g
Adjust pH to 7.5 with concentrated HCl, then dilute to desired volume
K.
Blocking Solution
Approximately 25 mls per membrane is required (dependent on the container
size). Make a solution of Tris buffered saline (TBS) with 0.1% tween-20 (100 ml
is a good volume). Take this solution and to make a solution with 5% dry milk.
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II.
Protein Gel Protocol
A.
Running Gel (10%)
3
For 24 ml (for 4 Gels)
8.0 ml
4.56 ml
3.0 ml
240 ul
12 ul
7 ml
Degas Solution
Ammonium Persulfate (100 mg/ml)
250 ul
Make fresh
30% Acrylamide
2.0M Tris-Cl pH 8.80
60% Sucrose
10% SDS
TEMED
dH2O
Can have all as stock except for Ammonium Persulfate which must be made up fresh
each time.
Two small gels only require 12 ml of above (actually 3.3 ml per gel)
Combine all of the above minus the APS and then degas for approximately 10-15
minutes.
Pour gel between plates (3.3 ml per gel), overlay with water and allow to polymerize for
approximately 0.75 – 1 hour.
B.
Stacking Gel (4.5%)
For 10 ml (for 2-4 gels)
30% Acrylamide
1.5 ml
0.5 M Tris-Cl pH 6.5
1.2 ml
10% SDS
100 ul
TEMED
5 ul
dH2O
7 ml
Degas Solution
Ammonium Persulfate (100 mg/ml)
200 ul
Make fresh
Combine all of the above minus the APS and then degas for approximately 10-15
minutes.
Pour gel between plates, insert comb between plates and allow to polymerize for
approximately 45 minutes to 1 hour.
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C.
4
Loading Gel (See Notes)
Load 5ul of molecular weight marker into the first lane. Load volume of SDS samples
into each lane to get 20ug per lane for rat tissues and 60-80ug for mouse heart Western
analysis. For Coomassie staining 10-15 ug is fine. For mouse brain load 5-10 ug
depending on antibody used.
C.
Running the Gel
1. Running the Gel (Using Electrophoresis Buffer)
The gel is run at 15 milliamps (constant current) until the samples are into the stacking
gel (approximately 15-20 min) at which time the current can be increased to 25-35
milliamps. The gel is run until the dye front just leaves the gel (approximately 1-1.5
hours).
D.
Staining the gel.
1. Commassie Staining:
The gel is removed from the gel apparatus and placed into a dish containing coomassie
stain. The dish is placed on a rocker and the gel is allowed to stain for at least 1 hour –
duration dependent upon total protein (overnight is okay). The gel is then transferred to
destaining solution with periodic solution changes with constant agitation. The gel can
then be viewed and photographed.
2. Immunoblots
Using the semi-dry blot transfer apparatus, cut a piece of filter paper large enough to fit
the gels being transferred. Wet the filter paper with blotting buffer and place on the
apparatus electrode, rolling it flat to remove any air bubbles. Cut a piece of transfer
membrane to fit the gel(s), wet it with blotting buffer and place on the filter paper. Place
the gel on top of the membrane and cover with another piece of wet filter paper. Be sure
to remove air bubbles at each level by rolling over it with a tube etc.
Place the cover on the apparatus and run the gel at 300 mA for 20 minutes per gel (linear
relation between time and number of gels). Remove the filter paper, gel and membrane
and place the membrane in blocking buffer overnight at 4°C with agitation.
3. Antibody Staining(See Notes)
Primary antibody is diluted in TBS-tween20 (TBST) solution (0.1% tween-20). For
PKC-epsilon (Santa Cruz) dilute 1:1000 (dave) or 1:200 (paul) or 1:2K-3.5K (brian –
mouse heart). Place membrane with protein in “seal-a-meal’ bag and add approximately
5ml (enough to cover). Seal bag and place on rocker for 1-2 hour at room temperature.
Wash membrane in dish with TBST solution for 25 minutes with aggitation at room
temperature. Change the solution several times.
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Horseraddish Peroxidase Secondary Antibody (IgG) stock is diluted to1:10K-20K in
TBST. 25ml is added to each gel in a dish and incubated for 1 hour at room temperature
with aggitation.
Wash membrane in dish with TBST for 25 minutes with aggitation at room temperature.
Solution should be changed several times.
4. Chemiluminesence Staining
Using an intensifier screen, cover with plastic wrap on to which the membrane will be
added and then covered (this protects the screen from the chemiluminescence chemicals.
Equal volumes of both reagents (company?) are added to a 50ml tube – approximately 10
ml of each per membrane. Solution can be poured between two dishes for approximately
2-10 minutes (depending on kit) covering the gels.
The membranes are then removed from the solution and placed on the plastic wrap on the
screen. The gels are then covered with the remaining plastic. A luminescence ruler is
placed at the top of the gel for alignment.
Expose the marker ruler (not usually membrane) to bright light for approximately 20 sec.
Turn off the lights and turn on the safety light before removing the unexposed film.
Place a piece of film over the gels (on top of the plastic), close the screen cover, and
allow to expose for a period of time (30 s – overnight). Open the screen, remove the film
and develop.
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NOTES
Note - Example Protein Weights:
PKC-epsilon
87-90
RACK
36
TnI
30
Tm
34
MLC I
25
MLC II
20
Tissue
Mouse Heart
Mouse Brain
Mouse Liver
Rat Heart
Rat Brain
Rat Liver
Rat Spleen
Protein Observed by Western Analysis (BBR)
PKC-ßI
PKC-ßII
PKC-
PKC-
X
X (C)
X
X
PKC-
X (C&P)
RACK I
X
X
X
X
X
Notes on Santa Cruz PKC Polyclonal Antibody Usage in Mouse Tissues
Here are suggested running concentrations
Antibody
Protein Per
Primary
Secondary
Lane (ug)
Dilution
Dilution
PKC-ßII
1-3
2K-10K
10K-20K
10-20
2K-10K
10K-20K
PKC-
C, P
C, P
Heart
PKC-ßII
PKC-
70-80
65-75
2K-10K
2K-10K
10K-20K
10K-20K
C, P
P
Liver
PKC-
70-80
2K-10K
10-20K
Tissue
Brain
* C-cytoplasmic fraction and P-particulate fraction
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Location*
P