发信人: nuonuo (nuo), 信区: Biology
标 题: Re: IP 求助！
发信站: BBS 未名空间站 (Wed Jan 30 20:54:06 2008)


【 在 juicemaker (Almost there) 的大作中提到: 】

: 2. How many total cellular protein do people use for IP? Or what is the
: rough efficiency of pulldown (say can 50% or more antigen could be
: ...................

My protocol (and it always works fine for me):
1. Non-denaturing lysis buffer, used for antigens that are detergent soluble
and can be recognised in native form by the antibody.
20 mM Tris HCl pH 8
137 mM NaCl
10% glycerol
1% Triton x-100
2 mM EDTA
store at 4oC
immediately before use add protease inhibitors

2. 10-500ug cell lysate. I routinely use ~200ug. you can check the antibody
datasheet for recommended antibody concentration. I usually use:
5 µl polyclonal antiserum
1 µl monoclonal antibody

let me know if u have more questions.

good luck!
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