SDS-polyacrylamide (SDS-PAGE)

50% Acrylamide/Bis (29:1) Solution

• 48.3 g acrylamide
• 1.7 g bis-acrylamide
• Make to 100 mL with water
• Store in a dark bottle.

Separating Gel Buffer (1 M Tris-HCl, pH 8.8)

• 30.3 g Tris
• Add 150 mL water and adjust to pH 8.8 with HCl.
• Make to 250 mL with water.

10% SDS

• 10.0 g SDS
• Make to 100 mL with water.

Stacking Gel Buffer (0.375 M Tris-HCl, pH 6.8)

• 11.4 g Tris
• Add 150 mL water and adjust to pH 6.8 with HCl
• Make to 250 mL with water.

10% Ammonium Persulfate

• 1 g ammonium persulfate
• Dissolve in 10 mL water.
• Aliquot and store at –20 °C.

Recipe for 12% separating gel

Makes 25 mL, enough for four 1.0 mm minigels.

• 6.0 mL 50% Acrylamide/Bis (29:1) solution
• 9.4 mL Separating Gel Buffer (1 M Tris-HCl, pH 8.8)
• 250 uL 10% SDS solution
• 9.0 mL water
• 20 uL TEMED¹
• 300 uL 10% ammonium persulfate¹

Notes

1. Add just prior to polymerization—open time is 5-10 min, full polymerization in 1 hr; layer with deionized water to prevent inhibition of polymerization by oxygen in air.

Recipe for 4% stacking gel

Makes 12.5 mL, enough for four 1.0 mm minigels.

• 1.0 mL 50% Acrylamide/Bis (29:1) solution
• 4.2 mL Stacking Gel Buffer (0.375 M Tris-HCl, pH 6.8)
• 125 uL 10% SDS solution
• 6.7 mL water
• 20 uL TEMED¹
• 500 uL 10% ammonium persulfate¹

Notes

1. Add just prior to polymerization—open time is 5-10 min, full polymerization in 1 hr; insert comb immediately after pouring..

Running Buffer (10 x)

• 29.0 g Tris
• 144.0 g glycine
• 10.0 g SDS
• Make to 1000 mL with water.
• Do not adjust pH

Loading Buffer (2 x)

• 2.5 mL 0.5 M Tris-Cl, pH 6.8
• 2.0 mL glycerol
• 4.0 mL 10% SDS
• 0.5 mL 0.1 % bromophenol blue
• 0.5 mL β-mercaptoethanol
• Make to 10 mL with water
• Aliquot and store at –20 deg C.

Running Conditions:

250 V constant, expect 60-80 mA per gel at start, 15-25 mA per gel at end. Total run time is approximately 45 min.

Low Toxicity Staining Solution

• 0.25g Coomassie Blue R-250
• 100 mL ethanol
• 100 mL water
• Stir until dye is dissolved
• Add 25 mL acetic acid and make to 250 mL with water
• Store at room temperature in a dark bottle.

Low Toxicity Destaining Solution

• 400 mL ethanol
• 100 mL acetic acid
• Make to 1000 mL with water
• Store at room temperature