发信人: reer (IE 疯了), 信区: Biology
标 题: Re: GST fusion protein-Thanks, buddy, but you guys misunderstood
发信站: The unknown SPACE (Sun Jan 19 19:20:52 2003) WWW-POST

1. maybe the problem is induction.
since i don't know the induction condition you use, here is my induction
condition for the protein I tried.(23 du, 2-3hrs) if still not good, try
18 du.
The proteolysis is inherent in GST fusion protein preps. You always get a 30
Kda form which is GST. The other problem about GST is that it is easy to form
dimer, especially the protein you work on is also easy to form dimer.
2. If you can not detect your protein in lysate, try to seqence your whole
vector. I know it is pain in a butt and a little bit ridiculous. But it really
happend to my labmate who sequenced the plasmid and found wrong sequence in
the promoter area.


【 在 Geneporter (Geneporter) 的大作中提到: 】
: What I am doing is western blot using bacteria lysate, directly lysed by
: adding SDS sample buffer and boiling for 5 minute. The lysis process only
took
: at most 30 seconds, there is no reason the GST fusion protein can be
degraded
: at this time. If it is degraded, it must be happened while its synthesis in
: the bacteria. Are there any protease inhibitor that I can apply while
: inducing?
:
:

--
杀，杀，杀
啥？啥？啥？
傻，傻，傻
煞！煞！煞！

※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 129.111.]