Thursday, October 05, 2006
Western Blotting
一直以為, western 是非常簡單的事, 總是用過去前人教我的方式做, Primary AB. overnight 4oC, wash 2x by TBST, secondary AB. 2-4 hours, wash 2X by TBST... 但是就是因為已為簡單的東西, 反而忽略了其一開紿的原理.
"Binding doesn't have activation energy"... Leroy不斷的提醒我們...
所以呢? 為什麼要4oc overnight? 不要已為不這樣子做會發生什麼怪事, 不要被perception所困惑. 要依循你的理智.
我的protocol: 從transfer開始..
1. rinse BA3 membrane in transfer buffer (with methanol)
2. package your stuff in normal running buffer ( you don't need to use transfer buffer to do this job)
3. run 400mA for 15 min, then maintain 400-450mA for 1hour( definitely enough, don't need to run longer)
4. wash your membrane with ddH2O, then rinse with Ponceus staining. (you will see very distinct and sharp band all over the membrance...smile!)
5. 5% milk for 40min-1hour...
6. Primary AB. room tempt. 1hour... don't shake it too hard! it is in equilibrium status.
7. wash by TBST 2X 15 min... don't shake it too mild!!!! don't be afraid to shake off your signal, it has already there.
8. Seconary AB.. please use 1:10.000 dilution.. that is right. you don't need to do anything tricky here, there is no mystery.... room tempt. < 1hour.
9. Wash by TBST 2X-3X 15 min... again,, just shake the fuck off it.
10. Use your homemade ECL reagent... get the most significant data.
1mM Luminol/ 8mM iodophenol???
35.5 mM H2O2
50mM glycine pH 9.6
"Binding doesn't have activation energy"... Leroy不斷的提醒我們...
所以呢? 為什麼要4oc overnight? 不要已為不這樣子做會發生什麼怪事, 不要被perception所困惑. 要依循你的理智.
我的protocol: 從transfer開始..
1. rinse BA3 membrane in transfer buffer (with methanol)
2. package your stuff in normal running buffer ( you don't need to use transfer buffer to do this job)
3. run 400mA for 15 min, then maintain 400-450mA for 1hour( definitely enough, don't need to run longer)
4. wash your membrane with ddH2O, then rinse with Ponceus staining. (you will see very distinct and sharp band all over the membrance...smile!)
5. 5% milk for 40min-1hour...
6. Primary AB. room tempt. 1hour... don't shake it too hard! it is in equilibrium status.
7. wash by TBST 2X 15 min... don't shake it too mild!!!! don't be afraid to shake off your signal, it has already there.
8. Seconary AB.. please use 1:10.000 dilution.. that is right. you don't need to do anything tricky here, there is no mystery.... room tempt. < 1hour.
9. Wash by TBST 2X-3X 15 min... again,, just shake the fuck off it.
10. Use your homemade ECL reagent... get the most significant data.
1mM Luminol/ 8mM iodophenol???
35.5 mM H2O2
50mM glycine pH 9.6
