DRL Lab, 1-3
Polyacrylamide Gel Electrophoresis (PAGE)
Stock Solutions:
A. Acrylamide/Methylene-bis-acrylamide:
a. Dissolve 30.0g acrylamide and 0.8g methylene-bis-acrylamide in about
80ml ddH2O.
b. Bring to 100ml with ddH2O
c. Store protected from light at ambient temperature.
B. Resolving Gel Buffer Stock (3.0 M Tris-HCl pH 8.8):
a. Dissolve 36.3g Trizma base in about 40ml 1.0N HCl.
b. Adjust pH to 8.8 with 6N HCl
c. Bring volume to 100ml with ddH2O.
d. Store at ambient temperature.
C. Stacking Gel Buffer Stock (0.5 M Tris-HCl pH 6.8):
a. Dissolve 3.0g Trizma base in 50ml ddH2O.
b. Adjust pH to 6.8 with 6N HCl.
c. Bring volume to 100ml with ddH2O.
d. Store at ambient temperature.
D. 50%(v/v) Glycerol:
a. Combine 50ml glycerol and 50ml ddH2O, and mix well.
b. Store at 4oC.
E. 10%(w/v) N-lauroylsarkosine:
a. Dissolve 10.0g N-lauroylsarkosine in about 80ml ddH2O.
b. Adjust pH to about 8.0 if necessary.
c. Bring volume to 100ml with ddH2O.
d. Store at ambient temperature.
F. 10%(w/v) Sodiuem dodecyl sulfate:
a. Dissolve 10.0g SDS in 80ml ddH2O.
b. Bring volume to 100ml with ddH2O.
c. Store at ambient temperature.
G. 10% (w/v) Ammonium Persulfate:
a. Dissolve 100.0mg ammonium persulfate in 1.0ml ddH2O.
b. Make this solution fresh each day.
Gel Preparation Recipes:
Preparation of Native Polyacrylamide Gel Electrophoresis (ND-PAGE) Stacking Gels:
Stock Solution
Volume of reagent needed for %T (ml)
(%T)
5% 6% 7% 8% 9% 10% 11% 12% 13% 14%
0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63
Stacking Gel Buffer Stock
0.83 1.00 1.17 1.33 1.50 1.67 1.83 2.00 2.17 2.33
Acrylamide/Bis
0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
50% Glycerol
0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
10% N-lauroylsarcosine
2.79 2.63 2.63 2.29 2.13 1.96 1.79 1.63 1.46 1.29
ddH2O
Recipes will make 5.0ml of monomer solution for one stacking gel
Preparation of Native Polyacrylamide Gel Electrophoresis (ND-PAGE) Resolving Gels:
15%
0.63
2.50
0.50
0.25
1.13
DRL Lab, 2-3
Stock Solution
Volume of reagent needed for %T (ml)
(%T)
5% 6% 7% 8% 9% 10% 11% 12% 13% 14% 15%
1.26
1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26
Resolving Gel Buffer Stock
1.66 2.00 2.34 2.66 3.00 3.34 3.66 4.00 4.34 4.66 5.00
Acrylamide/Bis
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
50% Glycerol
0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
10% N-lauroylsarcosine
5.58 5.24 4.90 4.58 4.24 3.90
3.58 3.24 2.90 2.58 2.24
ddH2O
Recipes will make 10.0ml of monomer solution for one resolving gel
Preparation of Denaturing Sodium Docecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) Stacking Gels:
Stock Solution
Volume of reagent needed for %T (ml)
(%T)
5% 6% 7% 8% 9% 10% 11% 12% 13% 14%
0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63
Stacking Gel Buffer Stock
0.83 1.00 1.17 1.33 1.50 1.67 1.83 2.00 2.17 2.33
Acrylamide/Bis
0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
10% SDS
3.44 3.28 3.11 2.94 2.78 2.61 2.44 2.28 2.11 1.94
ddH2O
Recipes will make 5.0ml of monomer solution for one stacking gel
15%
0.63
2.50
0.10
1.78
Preparation of Denaturing Sodium Docecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) Resolving Gels:
Stock Solution
Volume of reagent needed for %T (ml)
(%T)
5% 6% 7% 8% 9% 10% 11% 12% 13% 14% 15%
Resolving Gel Buffer Stock 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26
1.66 2.00 2.34 2.66 3.00 3.34 3.66 4.00 4.34 4.66 5.00
Acrylamide/Bis
0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20
10% SDS
6.88 6.54 6.20 5.88 5.54 5.20
4.88 4.54 4.20 3.88 3.54
ddH2O
Recipes will make 10.0ml of monomer solution for one resolving gel
Method:
1. Clean glass plates rigorously with ethanol before assembling gel sandwich.
2. Combine resolving gel reagents in glass beaker, add catalysts
a. 10% Ammonium Persulfate at 100ul/10ml gel
b. TEMED at 4ul/10ml resolving gel
3. Quickly pour gel, cover with milli-Q water, and allow to polymerize.
a. Allow resolving gels to polymerize about 1 hour
4. Pour off water and be sure that gel has a level top edge.
5. Combine stacking gel reagents in glass beaker, add catalysts
a. 10% Ammonium Persulfate at 50ul/5ml gel
b. TEMED at 5ul/5ml stacking gel
6. Quickly pour gel and allow to polymerize.
a. Allow staking gels to polymerize about 30 minutes.
7. Carefully remove comb(s) and rinse wells with Milli-Q water
8. Assemble gel box, attaching gel(s) to voltage carrier
a. If running only 1 gel use the buffer block on other side
DRL Lab, 3-3
9. Fill tank to the top of the plates with Running Buffer
10. Mix protein sample with sample buffer
a. For denaturing SDS gels use sample buffer with SDS and heat in boiling
water for 5 minutes to denature.
11. Load samples and molecular weight marker into gels and run at 130-150 V until
tracking dye is at bottom of gel.
12. Stain for enzyme activity and for total protein using standard methods.