发信人: moso (野鬼), 信区: Biology
标 题: Re: CoIP生手请教，急
发信站: BBS 未名空间站 (Tue Aug 30 23:20:39 2005), 转信

Before you can do anything to optimize your protocol, you'd better make sure
that all the reagents are correct.

For this particular case, I suppose the suspect band is neither your target
protein nor light/heavy chain , it either comes from your lysate, or protein A

beads, even your antibody. So,

1. Clean your beads before add them to your samples. Run a SDS-
PAGE to make sure that the beads are clean.

2. to make a preclear lysate is neccessary for you to rule out one of
the possibilities that there is non-specific protein interation between your
sample and beads.

3. Set another control. Mix only antibody and beads to see if it comes again
of
the suspect band.

4. You may need a more stringent washing condition if all above controls did
not give you any clue.

5. You need more luck. :)

【 在 cola (不要和陌生人说话) 的大作中提到: 】
: 我总是在Ig Control和Ab IP两个lane中得到同样大的被CoIP下来的蛋白带。倒是没有
替
: 他unspecific的带。
: 请问怎么才能让control干净些。
: 加抗体之前没有preclear，这个蛋白这么sticky怕在preclear中就弄没了
: 用的protein A的bead，因为抗体是Rb polyclonal
: lysis buffer是1%TX-100.
: 洗beads的时候洗很多次，vortex很长时间（15秒）
: 请问到底是那步的问题，怎么改进？



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