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

1. Spin down 100µL culture and suspend into sample buffer.
2. Boil at 95°C for 10 min.
3. Apply 10µL to polyacrylamide gel.
4. Electrophorese at 500V, 30mA for 50 min.
5. Transfer at 50V, 100mA for 30 min.
6. Add blocking buffer to the membrane and incubate with shaking for 30 min. at room temperature.
7. Add appropriate primary antibody diluted in TBST and incubate with shaking for 30 min. at RT.
8. Wash with TBST and incubate with shaking for 10 min, two times.
9. Add secondary antibody conjugated with AP and incubate with shaking for 30 min. at RT.
10. Wash with TBST and incubate with shaking for 10 min, three times.
11. Add 66µL NBT and 33µLof BCIP into color development buffer., and add the mixture to the membrane.
12.After the coloring, wash with DW.

Team:Kyoto/Protocol

From 2012.igem.org

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