发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: western blot问题
发信站: The unknown SPACE (Tue Nov 26 20:39:30 2002), 站内信件

you should set constant VOLTAGE instead of CURRENT.
i use semidry from BioRad, and it works very nice for my
western. set to 15V, transfer @ RT for 1hr to 1.5hr. i never
think that all the protein could be transferred onto the
membrane. and thus to make a homogenous sandwitch is very
important, which means that you treat the gel, the membrane,
as well as the blotting paper very equally well.
think about this, it is the electric field that pushes the
charged molecules to move rather than the current. if the
protein already transfer onto membrane (PVDF or Hybond ECL,
either one works for me), why should you still set very high
current? that will cause a lot of heat to melt the gel and
thus affect your blot. for a single blot 6cmX9cm, the current
@ 15V is about 150mA for me, and it drops very fast in 10min,
which means that the molecules that can move very fast has
already transferred. do a Ponceus S staining after transfer,
and acetic acid can help fix the protein.
actually, the transfer buffer i use contains 40mM glycine/
120mM glycine/0.04% SDS/15% methanol. remember that SDS will
cause heat, and an associate in my lab does not even use it.
for methanol, it is said that when the protein is large, you
can reduce it to 10%, otherwise, increase to 20%. i have used
this buffer to look at protein from 140kD to 20kD, and it
works. make 10X, add SDS/methanol when you use 1X, put it @
-20C before use. equilibrate the gel for 10minX2 @ 4C, that
helps prevent diffusion. do not put too much buffer onto the
blotting paper, and do not make it too dry either.


【 在 special (xxxx) 的大作中提到: 】
: 我用半干转移膜，200V20mA,但开始转移不久，电流就急剧下降，转移效果非常不好，我
: 敢肯定不是三明治短路造成的，因为我每次都特别注意。大虾们给出个主意。。


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※ 修改:．Marble 于 Nov 29 15:19:12 修改本文．[FROM: 128.118.]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.118.]