SDS-polyacrylamide gel electrophoresis


SOLUTIONS
30% acrylamide mix (30:0.8 bis) 30% (w/v) acrylamide
0.8% (w/v) N,N'- methylenebisacrylamide
5x Electrophoresis buffer Tris base: 15.1 g
Glycice: 94 g
SDS: 5 g
H2O 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%
H2O 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%
H2O 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)
H2O 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
H2O 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

20% methanol, 3% glycerol in diltilled water


Reference

Hames, D.(1990), One-dimensional polyacrylamide gel electrophoresis. In Hames, B.D. and Rickwood, D. (eds), Gel Electrophoresis of Proteins: A Practical Approach, 2nd ed., IRL Press, Oxford.




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