Polyacrylamide Gel System For Electrophoresis Of Proteins
Prepare 30.0% Acrylamide stock solution (30.0:0.8).
Prepare Resolving gel buffer (4X).
Prepare Stacking gel buffer (4X).
Prepare Running gel buffer (10X).
Prepare Cracking buffer, 4X (10 mL).
See guidelines for Sample Recipe for Resolving Gel.
Mix all of the ingredients together except the ammonium persulfate.
Gently overlay the gel with d-H2O (or 1X resolving gel buffer) and allow the gel to polymerize.
Pour the stacking gel immediately after adding the ammonium persulfate.
Insert the gel comb to form the wells.
Allow the stacking gel to polymerize for 60 min before use.
See guidelines for ingredients.
Boil the samples in the cracking buffer 5-10 min before loading onto the gel.
Electrophorese the gel at 100 volts (constant) or 10 milliamps (constant) until the dye front just runs off the gel.
See guidelines for Resolving Gel Recipes and Stacking Gel Recipes.
Stain the gels with coomassie blue stain according to the following recipe: 1.0 gm, Coomassie Brilliant Blue; R455 mL, methanol; 455 mL, d-H2O; 90 mL, glacial acetic acid;
Gels are destained with the following: 700 mL, methanol; 200 mL, glacial acetic acid; 2600 mL, d-H2O Stain and destain the gels at 65°C.
Small foam rubber pieces added during the destaining process absorb some of the coomassie with the result that less destain is needed and the destain need not be changed as often.
See guidelines for ingredients.
