Recovery of bands from SDS gels for MS

Ackerman, Eric J (eric.ackerman@pnl.gov)
Tue, 26 Jan 1999 17:42:32 -0800

We are trying to map partial proteolysis products of a recombinant
protein using mass spectrometry. When we directly analyzed the proteolysis
digest mixture by ms, we only found a subset of the products that should be
present. (We know there are missing fragments because SDS gel analysis clearly
reveals their presence.)

We'd like to excise bands from the SDS gel and analyze the recovered
fragments by ms. We have lots of the recombinant protein, so losses in the
extraction procedure are not a problem.

(1) Will Coomassie staining interfere with ms? If so, is there an alternative
stain suitable for subsequent ms?

(2) What is a good protocol to recover fragments from 10 to 30 kDa from SDS gels
for ms?

Thanks.

Eric Ackerman
Pacific Northwest National Laboratory
P.O. Box 999
Richland, WA 99352