Formaldehyde crosslinking - Compatibility with Mass spec (Oct/10/2005 )
I would like to study protein-protein inteactions by in vivo crosslinking using formaldehyde. I was considering the usual ChIP protocol steps of using 1% HCHO for 10-15 mins, quench by adding glycine, immunoprecipitate and then reverse crosslinks. But unlike ChIP, I want to analyze the interacting proteins using Mass spec.
When I talked to the staff in my Univ mass spec facility, they were concerned that formaldehyde crosslinks might cause some irreversible damage to proteins. I know that DNA-protein crosslinking is "reversible" for purposes of studying DNA... but does anyone know if protein-protein crosslinks could be reversed easily (and the proteins could be analyzed by mass spec)??. Also, I'd be grateful if someone can point me to a protocol for this process.
Thanks very much!
Formaldehyde crosslink should be reversible. prior to SDS-PAGE running, heating at 95C will remove all crosslinks.
Is this really true??? I thought that aldehydes form amid bonds when reacting with primary amino groups, which shouldn't be removed by boiling.
If it is true, would this mean that you can do WB from fixed tissue, maybe even from older archived tissue samples?
Thanks in advance,