the method you should try is that of Cohen and Chait using Formic
Acid:Isopropanol:Water to elute whole proteins from copper stained gels, if
you've got plenty of protein, it works very nicely. The reference is:
Cohen, S. L. and B. T. Chait (1997). "Mass spectrometry of whole proteins
eluted from sodium dodecyl sulfate- polyacrylamide gel electrophoresis
gels." Anal Biochem 247(2): 257-67.
Good luck.....Ken
> 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
>
********************************
Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
***********************************