Griffitts:Gel recipes

Denaturing gels

Resolving gel (15%) (5 mL)

• 550 μL dH₂0
• 938×2 μL 1 M Tris pH 8.8
• 25 μL 20% sodium dodecyl sulfate (SDS)
• 833×3 μL 30% acrylamide (29:1)
• 50 μL 10% ammonium persulfate (APS)
• 3 μL tetramethylethylenediamine (TEMED; in fume hood)

Resolving gel (10%) (5 mL)

• 690×2 μL dH₂0
• 938×2 μL 1 M Tris pH 8.8
• μL 20% sodium dodecyl sulfate (SDS)
• 833×2 μL 30% acrylamide (29:1)
• 50 μL 10% ammonium persulfate (APS)
• 3 μL tetramethylethylenediamine (TEMED; in fume hood)

Stacking gel (4%) (3 mL)

• 725×3 μL dH₂0
• 375 μL 1 M Tris pH 6.8
• 15 μL 20% 20% sodium dodecyl sulfate (SDS)
• 400 μL 30% acrylamide (29:1)
• 30 μL 10% ammonium persulfate (APS)
• 5 μL tetramethylethylenediamine (TEMED; in fume hood)

Native gels

Native Resolving gel (10%) (5mL)

• 900×2 μL dH₂O
• 938×2 μL 1 M Tris pH 8.8
• 625×2 μL 40% acrylamide (29:1)
• 50 μL 10% ammonium persulfate (APS)
• 3 μL tetramethylethylenediamine (TEMED; in fume hood)
• Overlay with dH₂O

Native Stacking gel (4%) (3mL)

• 760×3 μL dH₂O
• 375 μL 1 M Tris pH 6.8
• 300 μL 40% acrylamide (29:1)
• 30 μL 10% ammonium persulfate (APS)
• 5 μL tetramethylethylenediamine (TEMED; in fume hood)

Notes

• This protocol will work only for proteins with acidic pK_i (4–6.8)
• This protocol is the same as SDS-PAGE but with no SDS, and different sample buffer
• Do not boil sample
• 2-mercaptoethanol may be omitted from sample buffer