Griffitts:Gel recipes
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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 pKi (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