发信人: dawnsong (little+bear), 信区: Biology
标 题: Re: Western Blot 的膜怎么保存？
发信站: Unknown Space - 未名空间 (Sat Mar 5 07:03:21 2005) WWW-POST

【 在 magnolia (斗争到底) 的大作中提到: 】
: 放在牛奶里block overnight at 4 degree是不是不太好？我有时这样做， 尤其是很多
胶
: 的时候。
: 本来想做一系列perfect western blot, i was very careful, 但是美好的愿望常常和
现
: 实有距离， 做出来signal不强， 不知道问题出在哪里， protein
: prep应该没有问题，ponseau S staining的结果很好，antibody也绝对没有问题。之前
我
: 用相同条件做过一个pilot experiment, 结果非常beautiful, 洋洋得意了一会儿。我
预
: 计至少我可以得到相似的signal. however... 真是气恼啊！唯二的不同是block in
milk
: overnight, and ran gel longer(probably 30min more).还有after incubating with
: secondary antibody and wash,是不是越快加substrate反应越好啊， 不知道是不是巧
合
: ， 通常我有很多胶， 而且我使用biorad chemidoc to catch the chemiluninesence,

: 一般是一个膜一个膜来， 好像后面的膜signal没有前面的强，不知道是不是放得太久
了
: 。western blot我也做了很多了，问题还是层出不穷，郁闷啊。请有经验的人多多指教
，
: 谢谢！

I personally believe that there is no difference whether you block at RT 1 hr
or 4C ON. The same for the length of 2nd Ab incubation and wash. However, the
timing to catch the ECL signal, which will lose after half an hour, is
critical. When I have to do many blots together, I will do the blots that
probed with good 1st Abs( which give strong signal) first, then do the ones
interacted with weaker Abs. Another option is, do ECL seperately, or at most
two blots at one time. This will be more time consuming, but you can get good
results. Also, I found that if I was not able to catch the signal strong
enough the first time, I could just re-do the ECL (just inmerge the blot with
freshed mixed ECL solutions for one min), and get stronger sigal--- but this
have to be done before the blot get dried out.
:
: 【 在 dawnsong (little+bear) 的大作中提到: 】
: : 【 在 cola (不要和陌生人说话) 的大作中提到: 】
: : : PVDF膜， 刚block完，抗体有点问题至少得下周一或二才能继续做，应该怎么处理
呢
: ？
: : 能
: : : 弄干吗，还是泡在TBS里？
: : :
: : :
: : Keep in TBST(0.1%Tween 20) and put at 4C. It's fine for several days.
: :
: : Before block, you can either dry it or keep in TBST. I never tried to dry
it
: : after blocking, don't know it's good or not.
: :
: :
:
:


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