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Team:Kyoto/Protocol
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=Protocol= | =Protocol= | ||
| + | ===Western blotting=== | ||
| + | =====Gel solution===== | ||
| + | {| class="wikitable" | ||
| + | !||-|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 | ||
| + | |||
{{Kyoto/footer}} | {{Kyoto/footer}} | ||
Revision as of 13:02, 19 September 2012
Contents |
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
