Nuclear extracts for ChIP? - (Oct/19/2005 )
I read a couple of papers where people had made crude nuclear extracts before doing ChIP. I have tried doing this myself but seem to end up with very little DNA. I am wondering if this was a stupid idea, is there some part of the nuclear extract protocol that somehow gets rid of the DNA? I thought it might be a good idea as it will reduce the complexity of the sample. Also, I wanted to try digesting the DNA with a specific restriction endonuclease (to separate putative binding sites for my transcription factor within the promoter I'm studying) rather than sonication. Has anyone else tried this? I've done ChIP the 'traditional' way before and it worked ok but I did have a bit of trouble with high DNA backgrounds.
Your high DNA backgrounds are most likely a result of your sheering efficiency. At least in my experience I have found little difference between nuclear extract and whole cell extracts. It is easier to make the whole cell extract IMO, just optimize the conditions of your sheering ie sonication