crosslinking DNA to its binding protein invitro

Yuval Ebenstein via methods%40net.bio.net (by uv At chem.ucla.edu)
Sat Jan 6 18:10:25 EST 2007


Hi Again,
I've been working on this since my last post on the matter in October...
I'm trying to chemically crosslink the CAP (or CRP) DNA binding protein 
to a genomic fragment containing the binding site.
Gel shift confirms I have specific binding and a heparin challenge 
breaks the complex.
An extensive literature search yielded only invivo methods for chemical 
crosslinking, usually for CHIP by incubating with 1% formaldehyde.
However, doing the same invitro, and going up to 5% formaldehyde did not 
give me a complex that survived the heparin so i guess it is not 
crosslinked.
I might be doing something fundamentally wrong (there are no primary 
amines in my samples) or the efficiency is too low to detect in my case. 
I find it hard to believe that there are no protocols for this but I 
haven't found any. Anyway, i'm stuck!
Any help will be greatly appreciated
Cheers
Yuval

-- 
--------------------------------------------
Yuval Ebenstein
Department of Chemistry and Biochemistry, UCLA
Young Hall-2002, 607 Charles E. Young Drive East
Los Angeles, CA 90095-1569
Ph: (310) 794-6685; Fax: (310) 267-4672 



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