ChIP analysis - how to analyze CHIP data accuretly? - (Aug/04/2005 )
I wanted to know that what proper controls should be there in ur CHIP experiment. I included ANTIBODY negative only. But i saw in one paper using GAPDH (other than ur gene of interest, as a control) and then represented the data in relative to GAPDH.
I get band in no antibody control also. In that case using some internal control will be useful.
Can anyone enlighten me on this problem..
There will be a lot of ways to design your control.
A. use "input", which is the sample you obtained right after sonication.
B. use mock antibody, after sonication, divide your sample into two equal part, add your antibody/serum to one part, while add mock antibody (normal IgG) /pre-immune serum to the other.
OR, you must mainly follow a particular ChIP protocol, see what kind of control is used there.
Don't worry if you find control PCR product (e.g. GAPDH here) in mock ChIP sample. The most important thing you are looking for is the enrichment fold. For example, if you normalize GAPDH in both ChIP and mock ChIP DNA and you find PCR product of your candidate gene was significantly more in ChIP DNA, that mean there is enrichment.
Non-specific interaction always exist, no matter hwo hard you use washing buffer.