Griffitts:Gel recipes: Difference between revisions
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==Denaturing gels== | |||
===Resolving gel (15%) (5 mL)=== | ===Resolving gel (15%) (5 mL)=== | ||
* 550 μL dH<sub>2</sub>0 | * 550 μL dH<sub>2</sub>0 | ||
* 938×2 μL [[Griffitts:Common buffers#1 M Tris, pH 8.8 (75 mL)|1 M Tris pH 8.8]] | * 938×2 μL [[Griffitts:Common buffers#1 M Tris, pH 8.8 (75 mL)|1 M Tris pH 8.8]] | ||
* 25 μL [[Griffitts:Stock solutions#20% SDS (75 mL)|20% sodium dodecyl sulfate (SDS)]] | * 25 μL [[Griffitts:Stock solutions#20% SDS (75 mL)|20% sodium dodecyl sulfate (SDS)]] | ||
* 833×3 μL 30% acrylamide (29:1) | * 833×3 μL [[Griffitts:Stock solutions#30% Acrylamide (75 mL)|30% acrylamide]] (29:1) | ||
* 50 μL 10% ammonium persulfate (APS) | * 50 μL [[Griffitts:Stock solutions#10% Ammonium persulfate (75 mL)|10% ammonium persulfate (APS)]] | ||
* 3 μL tetramethylethylenediamine (TEMED; in fume hood) | * 3 μL tetramethylethylenediamine (TEMED; in fume hood) | ||
===Resolving gel (10%) (5 mL)=== | ===Resolving gel (10%) (5 mL)=== | ||
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* 938×2 μL [[Griffitts:Common buffers#1 M Tris, pH 8.8 (75 mL)|1 M Tris pH 8.8]] | * 938×2 μL [[Griffitts:Common buffers#1 M Tris, pH 8.8 (75 mL)|1 M Tris pH 8.8]] | ||
* μL [[Griffitts:Stock solutions#20% SDS (75 mL)|20% sodium dodecyl sulfate (SDS)]] | * μL [[Griffitts:Stock solutions#20% SDS (75 mL)|20% sodium dodecyl sulfate (SDS)]] | ||
* 833×2 μL 30% acrylamide (29:1) | * 833×2 μL [[Griffitts:Stock solutions#30% Acrylamide (75 mL)|30% acrylamide]] (29:1) | ||
* 50 μL 10% ammonium persulfate (APS) | * 50 μL [[Griffitts:Stock solutions#10% Ammonium persulfate (75 mL)|10% ammonium persulfate (APS)]] | ||
* 3 μL tetramethylethylenediamine (TEMED; in fume hood) | * 3 μL tetramethylethylenediamine (TEMED; in fume hood) | ||
===Stacking gel (4%) (3 mL)=== | ===Stacking gel (4%) (3 mL)=== | ||
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* 375 μL [[Griffitts:Common buffers#1 M Tris, pH 6.8 (75 mL)|1 M Tris pH 6.8]] | * 375 μL [[Griffitts:Common buffers#1 M Tris, pH 6.8 (75 mL)|1 M Tris pH 6.8]] | ||
* 15 μL 20% [[Griffitts:Stock solutions#20% SDS (75 mL)|20% sodium dodecyl sulfate (SDS)]] | * 15 μL 20% [[Griffitts:Stock solutions#20% SDS (75 mL)|20% sodium dodecyl sulfate (SDS)]] | ||
* 400 μL 30% | * 400 μL [[Griffitts:Stock solutions#30% Acrylamide (75 mL)|30% acrylamide]] (29:1) | ||
* 30 μL 10% ammonium persulfate (APS) | * 30 μL [[Griffitts:Stock solutions#10% Ammonium persulfate (75 mL)|10% ammonium persulfate (APS)]] | ||
* 5 μL tetramethylethylenediamine (TEMED; in fume hood) | * 5 μL tetramethylethylenediamine (TEMED; in fume hood) | ||
==Native gels== | |||
===Native Resolving gel (10%) (5mL)=== | |||
* 900×2 μL dH<sub>2</sub>O | |||
* 938×2 μL [[Griffitts:Common buffers#1 M Tris, pH 8.8 (75 mL)|1 M Tris pH 8.8]] | |||
* 625×2 μL [[Griffitts:Stock solutions#40% Acrylamide (75 mL)|40% acrylamide]] (29:1) | |||
* 50 μL [[Griffitts:Stock solutions#10% Ammonium persulfate (75 mL)|10% ammonium persulfate (APS)]] | |||
* 3 μL tetramethylethylenediamine (TEMED; in fume hood) | |||
* Overlay with dH<sub>2</sub>O | |||
===Native Stacking gel (4%) (3mL)=== | |||
* 760×3 μL dH<sub>2</sub>O | |||
* 375 μL [[Griffitts:Common buffers#1 M Tris, pH 6.8 (75 mL)|1 M Tris pH 6.8]] | |||
* 300 μL [[Griffitts:Stock solutions#40% Acrylamide (75 mL)|40% acrylamide]] (29:1) | |||
* 30 μL [[Griffitts:Stock solutions#10% Ammonium persulfate (75 mL)|10% ammonium persulfate (APS)]] | |||
* 5 μL tetramethylethylenediamine (TEMED; in fume hood) | |||
===Notes=== | |||
*This protocol will work only for proteins with acidic pK<sub>i</sub> (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 | |||
Latest revision as of 13:56, 11 April 2008
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
