f***r 发帖数: 204 | 1 还在与PVDF战斗中,再次请教有经验的虾们:
终于看到了条带,但是巨多非特异带,比我要的带都强得多。在网上查到多家western系
统的trouble shooting,里面提到的好几条关于high background的建议让我大开眼界,
又困惑非常,因为矛盾很多:
1. 关于tween的使用:有一家建议blocking之后才用tween,否则tween会stick to the
membrane and cause high noise;但很多常规方法都没有提到这一点。
2. 关于blocking reagents和ab incubation buffer:有说dry milk能reduce
sensitivity,所以如果只用在二抗incubation就能很好降低noise,但也有很多全程用dr
y milk的,也有说dry milk与tween联合使用会提高noise的;有说BSA会造成高背景的,
也有说用BSA可以获得理想信噪比的……
3. 关于ponceau s染色:这次为确证transfer有效,用了ponceau s染色,但发现有资料
说ponceau s就算洗干净也会 | e**e 发帖数: 16 | 2
much
It does not matter if you use NC or PVDF although I like NC from S&S.
PS staining did give some background when it was not rinsed enough. If you use
heavily diluted PS and rinse with 10X TBS for short time, it is very easy to
get rid of it with no loss of protein.
Skim milk is working fine in most cases and cheap, so we love to use it and so
far no problems.
To get better specificity and less background, you may incubate with 1Ab as
less as it works at 4C o/n, also for the 2Ab. If you use
【在 f***r 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 还在与PVDF战斗中,再次请教有经验的虾们: : 终于看到了条带,但是巨多非特异带,比我要的带都强得多。在网上查到多家western系 : 统的trouble shooting,里面提到的好几条关于high background的建议让我大开眼界, : 又困惑非常,因为矛盾很多: : 1. 关于tween的使用:有一家建议blocking之后才用tween,否则tween会stick to the : membrane and cause high noise;但很多常规方法都没有提到这一点。 : 2. 关于blocking reagents和ab incubation buffer:有说dry milk能reduce : sensitivity,所以如果只用在二抗incubation就能很好降低noise,但也有很多全程用dr : y milk的,也有说dry milk与tween联合使用会提高noise的;有说BSA会造成高背景的, : 也有说用BSA可以获得理想信噪比的……
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