ChIP analysis using tissue and controls - (Jan/04/2006 )
I am trying to use ChIP analysis with tissue from either mouse or monkey. However, in the past I have only done ChIP using tissue culture cells. Does anyone have a protocol for crosslinking and isolating cells from tissue for a ChIP experiment? How much tissue do I need?
Also, I have a question regarding what negative control to use. In the past I have using IgG as a negative control, but I've been told that could give me false results. Should I use no antibody, a non-specific antibody, etc.
Any help would be appreciated.
Sorry I can't help with crosslinking in a tissue but regarding your question about using mock IP for normalization I can say we use IgG or specific antibody blocked with it's peptide epitope for normalization/negative control. We do this because it has been demonstrated that chromatin IP has a bias in pulling down transcribed regions more than intergeneic regions. This would make normalizing with input DNA inappropriate (Cell 122, 517-527 (2005)). In any case, normalizing with input DNA or with mock ChIP (ChIP with IgG) is currently accepted in publications so you could probably go either way.
here is a helpful protocol for chromatin isolation from tissue
also, you can crosslink brain tissue in vivo by perfusing with 4% PFA, post-fixing, and then emersing tissue in 20% sucrose solution for 48hrs. (weaver ICG et al 2005)