发信人: OzO (jjjjjjjj), 信区: Biology
标 题: Re: 有人对CO-IP比较熟嘛？？
发信站: BBS 未名空间站 (Mon Sep 25 02:02:02 2006), 转信

你的操作看起来大体没错。overexpress 相互作用，endogenous作不出来的例子很多。
除了你的两个protein真的不interact以外，一些可能的解释：
1. 你的anti－A antibody 正好react to A-B之间的interacting motif
2. the interaction may be transient or depends on other factors (eg,cell
cycle).
3. the protein interaction may be weak, so you need to adjust the
concentration of detergent in your IP buffers. for example，you can use
freeze-thaw to disrupt cells in very low percentage of detergent and perform
the IP thereafter.
最后，CO-IP是很难把握的实验，如果这个project对你不是很重要的话，也别押上太多
的时间。
【 在 IVYtony (村委书记) 的大作中提到: 】
: 最近刚开始试着做CO-IP，用的是SLB-1 cell line (HTLV-1 infected cells),试图
stu
: dy the in-vivo interaction between endogenous proteins A and B.
: A is unknow to over-express in SLB-1 cells and B was also detected in the
: cel
: l lysates of SLB-1. In addition, A interacts with B in vitro, which has
been
: previously observed by our lab. So I'm trying to detect the A-B
interaction
: in vivo.
: I started with CO-IP to analyze the A-B interaction. Basically, I first
obta
: in the whole cell extracts of SLB-1 cells (2X10^7 cells approx.). Before
car
: rying out CO-IP, I performed a western blot of the cell lysates using anti
-A
: ...................


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