|MadSci Network: Biochemistry|
Dear Supriya, I personally am not an expert on autoradiography or biochemistry of proteins, so I ask Dave, a biochemist colleague who does mass spectrometry. His answer is below... Hi Ken, Yes I am familiar with the technique in question. There are protocols out there that deal with 1-D and 2-D gels (which is probably how this protein was separated prior to binding to the membrane and exposing the film), but to my knowledge, not a lot out there on retrieving proteins from nitrocellulose membranes. I assume that cutting out a bit of the membrane would be easy enough, but since the proteins are typically fixed to the membrane, getting the intact protein back would be a challenge. You could do a trypsin digestion on the membrane spot and probably liberate some peptides (after a denaturing step). Typically when people do "MS" on a gel spot (or in this case, a membrane spot) they're looking to do peptide mass fingerprpinting to get the protein ID. That requires a proteolytic digestion anyway, so performing something like a trypsin digest on the membrane piece you've identified by autoradiography would seem reasonable. However, this all assumes that the owner of the mass spec doesn't mind you putting a radiolabeled protein in his/her instrument. I wouldn't allow it ;-). I hope this helps. Dave
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