Protocol
Western blotting
Gel solution
| Running gel | Stacking gel
|
1.5M Tris-HCl(pH8.8) | 2.5mL | -
|
0.25M Tris-HCl(pH6.8) | - | 3.0mL
|
30% Acrylamide | 5mL | 0.6mL
|
10% SDS | 0.2mL | 0.12mL
|
DW | 2.3mL | 2.3mL
|
TEMED | 15µL | 7µL
|
10% APS | 100µL | 60µL
|
Total | 10mL | 6mL
|
4x Sample buffer
1M Tris-HCl 2mL
SDS 0.8g
100% glycerol 4mL
14.7M mercaptoethanol 0.4mL
0.5M EDTA 1mL
Bromophenol blue 8.0mg
DW 2.6mL
10x electrode buffer
Tris 15.15g
Glycin 71.55g
10%SDS 50mL
DW 450mL
Total 500mL
blotting buffer
Tris 12g
Glycin 14.4g
DW 800mL
Methanol 200mL
TBST
50mM Tris
150mM NaCl
0.1% Tween-20
blocking buffer
TBST
5% skim milk
AP color development buffer
100mM Tris (pH8.5)
100mM NaCl
5mM MgCl2