Time required: Individual Steps: Pouring Separating Gel: 60 minutes Pouring Stacking Gel: 30 minutes Loading Samples 15 minutes Electrophoresis: Staining: Coomassie Staining: 30 minutes (major bands),3 hours for major bands to destain Complete destaining may require 24-48 hours. Silver staining: 6 hours. IL BUFFER SYSTEM (A): Lower buffer: 18.17gTis, 0.4g SDS, pH8.8(HCI), Added H2o to 100ml (1. 5M Tris-HCl buffer) (B): Upper buffer: 6.06g Tris, 0.4g SDS, pH 6.8(HCI), Added h2o to 100ml (0.5M Tris-HCl buffer (C): 30% Acrylamide 30g Acrylamide, 0.8g Bisacrylamide, Added H20 to 100ml (D): 10%(w/v)Ammonium persulfate(fresh): 0. 1g ammonium persulfate Added Ho to lml (F): Electrophoresis Buffer: 14.4g glycine, 1g SDS, Added H20 to 1 liter. *H2O: Distilled water *Amounts of working Solutions to Use196 Time Required: Individual Steps: Pouring Separating Gel: 60 minutes Pouring Stacking Gel: 30 minutes Loading Samples: 15 minutes Electrophoresis: 45 minutes Staining: Coomassie Staining: 30 minutes (major bands), 3 hours for major bands to destain. Complete destaining may require 24-48 hours. Silver Staining: 6 hours. II. BUFFER SYSTEM: (A): Lower buffer: 18.17g Tris, 0.4g SDS, pH 8.8 (HCl), Added H2O to 100ml (1.5M Tris-HCl buffer). (B): Upper buffer: 6.06g Tris, 0.4g SDS, pH 6.8 (HCl), Added H2O to 100ml (0.5M Tris-HCl buffer). (C): 30% Acrylamide: 30g Acrylamide, 0.8g Bisacrylamide, Added H2O to 100ml. (D): 10%(w/v) Ammonium persulfate (fresh): 0.1g ammonium persulfate, Added H2O to 1ml. (F): Electrophoresis Buffer: 3g Tris, 14.4g glycine, 1g SDS, Added H2O to 1 liter. *H2O: Distilled water. *Amounts of Working Solutions to Use