SDS-polyacrylamide gel electrophoresis

SOLUTIONS

30% acrylamide mix

5x Electrophoresis buffer

4x Resolving-gel buffer

8x Stacking-gel buffer

1x SDS gel-loading buffer

29% (w/v) acrylamide
1% (w/v) N,N''- methylenebisacrylamide
Tris base: 15.1 g
Glycice: 94 g
SDS: 5 g
H2 O upto1 liter
1.5M Tris-Cl (pH 8.8)
1M Tris-Cl (pH 6.8)
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

30% acrylamide mix

4x resolving-gel buffer

20% SDS

20% APS

TEMED

16.2	14.2	12.2	10.2	7.2
6.0	8.0	10	12	15
7.5	7.5	7.5	7.5	7.5
0.15	0.15	0.15	0.15	0.15
0.15	0.15	0.15	0.15	0.15
0.024	0.018	0.012	0.012	0.012

Resolving Gel (10ml)6%8%10%12%15%H2 O

30% acrylamide mix

4x resolving-gel buffer

20% SDS

20% APS

TEMED

5.4	4.7	4.1	3.4	2.4
2.0	2.7	3.3	4	5
2.5	2.5	2.5	2.5	2.5
0.05	0.05	0.05	0.05	0.05
0.05	0.05	0.05	0.05	0.05
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

Destaining 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.
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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