SDS-polyacrylamide gel electrophoresis
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1502
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