SDS-polyacrylamide gel electrophoresis
SOLUTIONS30% acrylamide mix29% (w/v) acrylamide1% (w/v) N,N''- methylene
SOLUTIONS
| 30% acrylamide mix | 29% (w/v) acrylamide |
|---|---|
| 1% (w/v) N,N''- methylenebisacrylamide | |
| 5x Electrophoresis buffer | Tris base: 15.1 g |
| Glycice: 94 g | |
| SDS: 5 g | |
| H2 O upto1 liter | |
| 4x Resolving-gel buffer | 1.5M Tris-Cl (pH 8.8) |
| 8x Stacking-gel buffer | 1M Tris-Cl (pH 6.8) |
| 1x SDS gel-loading buffer | 50mM Tris-Cl (pH 6.8) |
| 100mM dithiothreitol | |
| 2% SDS | |
| 0.1% bromophenol blue | |
| 10% glycerol |
RESOLVING GEL MIXTURE
| Resolving Gel (30ml) | 6% | 8% | 10% | 12% | 15% |
|---|---|---|---|---|---|
| H2 O | 16.2 | 14.2 | 12.2 | 10.2 | 7.2 |
| 30% acrylamide mix | 6.0 | 8.0 | 10 | 12 | 15 |
| 4x resolving-gel buffer | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 |
| 20% SDS | 0.15 | 0.15 | 0.15 | 0.15 | 0.15 |
| 20% APS | 0.15 | 0.15 | 0.15 | 0.15 | 0.15 |
| TEMED | 0.024 | 0.018 | 0.012 | 0.012 | 0.012 |
| Resolving Gel (10ml) | 6% | 8% | 10% | 12% | 15% |
|---|---|---|---|---|---|
| H2 O | 5.4 | 4.7 | 4.1 | 3.4 | 2.4 |
| 30% acrylamide mix | 2.0 | 2.7 | 3.3 | 4 | 5 |
| 4x resolving-gel buffer | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
| 20% SDS | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 |
| 20% APS | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 |
| TEMED | 0.008 | 0.006 | 0.004 | 0.004 | 0.004 |
STACKING GEL MIXTURE
| Stacking Gel (10ml) | ||
|---|---|---|
| H2 O | 6.9 ml | |
| 30% acrylamide mix | 1.7 ml | |
| 8x stacking-gel buffer | 1.25 ml | |
| 20% SDS | 0.05 ml | |
| 20% APS | 0.05 ml | |
| TEMED | 0.01 ml |
SOLUTIONS FOR STAINING AND DESTAINING
| Staining solution | methanol | 90 ml |
|---|---|---|
| H2 O | 90 ml | |
| acetic acid | 20 ml | |
| Coomassie Brilliant Blue R250 | 0.25 g | |
| Dissolve well and filtrate through a filter paper. | ||
| Destaining solution | isopropanol | 12.5% |
| glacial acetic acid | 10% | |
| Addition of small pieces of used X-ray film with the gel will accelerate the destaining process. |
SOLUTION FOR DRYING
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