发信人: sunnyday (飞鸟), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Fri Oct 4 12:24:26 2002) WWW-POST

Just like other gus have already said:
1. Buy another antibody from another specie. (I highly recommend this way)

2. crosslink your antibody to gel particles (CNBr activated Sepharose),
use the gel to pull down your protein,then use high salt(eg., 8M urea) to
seperate your protein.
3. crosslink your antibody to your protein by EDC-NHS method, then your
target will have a higher molecular weight. However, the result can turn out
to be hard to interpreted since the antibody would also bind something else
although with lower affinity.

【 在 gardenia (缘了，就是完) 的大作中提到: 】
: the problem is my protein is around 55kDa
: the same as heavy chain of primary Ab
: and i always see a strong band there, i know it is the Ab,
: but i don't know whether my protein is also there
: 【 在 Marble (小石头哥哥) 的大作中提到: 】
: : your problem is not western. the key problem is that the
: : amount of the endogenous protein that you are interested
: : is so small that you could hardly detect it by common
: : immunoblot. you may try to use some hypersensitive film
: : for visulization, or enrich the protein as bongbongcat has
: : suggested. btw, i would rather do in situ hyb for RNA. how
: : much would like to know? is the western a necessity for
: : manuscript? i have seen many papers with no expression profile
: : of endogenous protein. and what they usually do is to over
: : express the protein in some cell lines that have very low
: : amount of such protein. but technically speaking, i would not
: : quite favor such method since it overloads the cell system with
: : enormous amount of protein. so could you specify your question
: : since for different proposal you have to use diff. approaches.
: : 【 在 bongbongcat (cutie) 的大作中提到: 】
: : : cross link your ab to the beads before adding it to the cell lysate.
this
: : : way, it will not be released from the beads with boiling in sample
buffer.
: : : good luck
:
:


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