formaldehyde crosslink question - (Nov/29/2005 )
Just 2 quick questions:
In ChIP, at any step before removal of protein (from DNA), is the protein denatured? I.e., is its structure intact through formaldehyde crosslinking and immunoppt?
Also, is it practical to use formaldehyde crosslinking to see if a protein X is bound to another protein Y while protein Y is bound to DNA?
Thanks in advance!
1) before removal of protein is the immunoprecipitation and elution, so I guess the protein should not be denatured. It it possible the elution at 65C in 1% SDS might denature some proteins... But some people do double immunoprecipitations, rediluting the eluate and adding some antibody (same or a different one), so it should not be denatured. Why do you ask?
2) I've seen some coactivators (cofactors) "Chipped" in the literature, so it should work. Or you could add a second crosslinking agent. Take a look a this paper:
Two-step cross-linking method for identification of NF-κB gene network by chromatin immunoprecipitation.
Hope this helps, good luck!
I was just wondering if it was practical to do some form of structural analysis even after immunoppt and all that. Although I guess most strucutral analysis requires the protein to be in solution and not precipitated out.
Thanks a bunch for the quick reply!