Ken Mitchelhill wrote:
> Eric,
>
> 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
>
> ***********************************