发信人: mnt (wait), 信区: Biology
标 题: Re: Need help please on WB
发信站: Unknown Space - 未名空间 (Wed Mar 31 00:46:21 2004) WWW-POST

Let me make a bold guess just for fun. :)))
Assume that the band you have seen after 2Ab is the correct band instead of a
crossreactivity artifact, I guess what was happening is that you 1Ab has a
very weak interaction against the fusion protein. The Ab can be easily washed
off in the subsequent washing steps, however, when you added destain solution,
the methanol fixes the 1Ab on the blot so that it woon't be totally washed.
Therefore the 2Ab can detect the 1Ab on the membrane, and that's also why you
get very high background.
If that is the case, several things can be done to improve the result without
using the destain. For instance, increasing sample loading, decreasing the
salt and detergent in your wash buffer, looking for a better 1Ab, using a more
sensitive substrate. etc.
【 在 Elizabeth (Ely) 的大作中提到: 】
: I have a very confusing situation here. I was trying to detect Gal4 fusion
: protein expression in a cell line. And through the regular protocol, I was
: never be able to detect anything. Then one day, when I wash the membrane
after
: primary antibody, accidentally, I poured destaining buffer (which has
methanol
: and acetic acid) onto the membrane. I washed and proceed with 2nd antibody.
: Although I got very dark background, I was able to see the fusion protein
: band. So why is this? I repeated several times, every time I destain, there
is
: my protein band, However, every time I don't do destain, no band! Please
help
: me, I'm kind of dying here.
:
:


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