Immunoprecipitation - (Jul/13/2007 )

Hi Guys,

I'm doing IP with a nuclear protein, used 250ug nuclear extract, incubate with primary antibody for 7hr at 4C, then incubate with protein A sepharose overnight at 4C. Did not get any band with western blot (the nuclear extract does get the band). Is 250ug of nuclear extract not enough? or is there any trick to do the nuclear IP?

Thanks in advance for your kind help.

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there may be several pitfalls: wrong buffer, too little primary Ab or inadequate (= non-suitable for IP) Ab, too little abundance of your protein of interest in the extract etc

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Hi Thanks for you reply. I think my buffer (RIPA) should be fine. 1st Ab should be OK, it was from cell signaling and the manual suggested 1:100 dilution for IP -- should I increase the 1st Ab, such as 1:50 dilution? I can get a very bright brand of my target protein with 30ug nuclear extract by western blot. I did another IP with 1mg of nuclear extract and got a very very weak band with western blot, but surprising another band (~70kDa bigger than my target protein) is much brighter. I just found out a paper, it used 2mg nuclear extract and got the results I want -- isn't 2mg nuclear extract too much?

Thanks for your time.

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