I am doing IP of my POI, a nuclear protein. Ac-POI is a kind of transcriptional factor. I have several questions.
1) How to protect acetylation modification of POI? Protease inhibitor cocktail alone is enough?
2) What lysis buffer is good to disrupt nuclear membrane and release nuclear protein WITHOUT interfering with subsequent POI-antibody binding?
3) For IP of my POI, total cell lysate is good for IP or nuclear lysate?
Hi colleague,
regarding question 1), I have no experience, but as a lysis buffer, I would suggest to use a hypotonic buffer with 0.5 to 1% NP-40, and in case this is not enough to disrupt the membrane, you can add up to 0.5% Triton-X-100 in your buffer without influencing antibody binding. 3) Definitely use a nuclear lysate which was properly homogenized and precleared before, for example following a method like this: http://www.nature.com/nprot/journal/v1/n4/full/nprot.2006.273.html
Hope this helps!
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