发信人: windysea (雨瞳~甜心小猪~babymaking), 信区: Biology
标 题: Re: nonspecific band
发信站: Unknown Space - 未名空间 (Mon Mar 28 18:54:41 2005) WWW-POST

Is your blot after IP or just straight Western - I guess it is straight
Western on whole lysate,
then, since the Ab can recognize this nonspecific band,
in theory it is very likely to pull down this protein.
so, you may do IP with this Ab (you should scale up!),
try Coomassie staining on SDS-PAGE, cut the band at the MW of your interest,
and then you can get the potein ID by MS (a band which is just visible is
enough).

However, since I assume you are talking about endogenous protien in your whole
lysate,
even if you scale up a great deal,
chances are you still don't get a visible band by Coomassie.
Then try silver staining,
whose disadvantage is that it seems to be less compatible to MS (at least in
our hands), but many ppl succeeded it in their publications.

in either case, before you cut the band, do compare the pattern with your
Western in which you observed 理想的变化, in order to make sure it is the same
band.

Now you can see that this approach is actually quite tough,
If I were you, I wouldn't do it unless I had reasons exciting enough.
I actually don't understand why you are interested in a non-specific band -
many nonspecific bands are just very abundant proteins;
neither do I understand what you meant by 发生理想的变化, can you tell me?

Also, see if other ppl have better approaches



【 在 raison () 的大作中提到: 】
: thanks a lot. can you talk a bit more in detail? i've never done that
:
: 【 在 windysea (雨瞳~甜心小猪~babymaking) 的大作中提到: 】
: : mass spec
: : 【 在 raison () 的大作中提到: 】
: : : 作western时， 发现一个nonspecific band发生理想的变化，只知道大概的分子量
，
: 有
: : 没
: : : 有办法搞清楚到底是什么PROTEIN呢？
: : :
: : :
: :
:
:



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※ 修改:·windysea 於 Mar 28 18:54:41 修改本文·[FROM: 140.163.]
※ 来源:．Unknown Space - 未名空间 mitbbs.com．[FROM: 140.163.]