MadSci Network: Biochemistry

Re: can we do mass spectrometry of the protein after doing autoradiography?

Date: Wed May 26 11:50:33 2004
Posted By: Kenneth Beck, Staff, Chemistry and Physics of Complex Systems (C&PCS), Pacific Northwest National Laboratory
Area of science: Biochemistry
ID: 1085500998.Bc

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.


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