发信人: Cajal (chump), 信区: Biology
标 题: Re: CoIP生手请教，急
发信站: BBS 未名空间站 (Wed Aug 31 17:04:37 2005)

We sometimes have this problem. These are what we would try when we are stuck:
1. make sure that the DNA in your lysate get totally removed. because the DNA
fragments in lysate can make the solution very sticky and tend to stick on
everything. You can do: a)shear DNA with 21G syringe many times, spin down the
debris for 10 min in bench top centrifuge. b)passing the lysate through QIA-
shredder from QIAgen before shearing with syringe can speed up the process. c)
If you are sure about the localiztion of your protein (cytoplasmic or nuclear)
, you can try the NE-PER nuclear extraction kit from Pierce, which allow you
to get both nuclear and cytoplasmic extract.

2. Instead of using rabbit serum or nonspecific IgG as control, you may try
using an irrelavent pAb from the company. For example, your experiment is IP
with poly-Anti-Rb, you may try poly-anti-GFP as control. This control should
be cleaner and less sticky.

3. Can you bypass the IP by doing an "in vivo" pulldown? Clone your Rb into a
pGEX vector and express the protein. Pre-bind the GST-Rb on glutathione beads,
then incubate the beads with lysate. Use GST protein alone as control. The
problem of this resort is that it may not give true result on the proteins
require phosphorylation; and sometimes the bulky GST tag may interfere with
protein-protein interaction.

【 在 cola (不要和陌生人说话) 的大作中提到: 】
: 那个带是我想要的蛋白，但是它在control里比CoIP的lane里还强。
: 你说的我都会试试，谢谢啦，尤其是最后一条，真的比什么都重要啊
: 作western真是个费劲费时间又不讨好的活，sigh
: 几个月过去了都做不出来一个能见人的结果
: A
:



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