发信人: else (where), 信区: Biology
标 题: Re: 大家再来个PVDF western大讨论好吗？
发信站: The unknown SPACE (Thu May 22 12:17:57 2003) WWW-POST

【 在 fever (葡式蛋塔) 的大作中提到: 】
: Please, could you give more details upon what should be done to avoid too
much
: background? Did you use dry milk or BSA in blocking solution? Did you use
: tween-20 through the detection steps? //bow

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 AP conjugated, be
sure to prepare the substrate buffer at pH9.5 around and incubate in dark; if
you use ECL, ask people around to give you tips on exposure time.
Through whole the procedure, incubation buffer always has 0.01~0.1% T20, which
is exempted from washing buffer.
Use TBS rather than PBS.
Above I am talking about the non-specific staining generated by hands. If your
protein itself has degradation or whatever happened, forget the reply. Also
check if the Abs are ok.


--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 130.60.]