Team:Kyoto/Protocol

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Contents

Protocol

Western blotting

Gel solution
Running gelStacking gel
1.5M Tris-HCl(pH8.8)2.5mL-
0.25M Tris-HCl(pH6.8)-3.0mL
30% Acrylamide5mL0.6mL
10% SDS0.2mL0.12mL
DW2.3mL2.3mL
TEMED15µL7µL
10% APS100µL60µL
Total10mL6mL
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