Rebecca,
Looks like most of the aspects of in situ digest (or cleavage) with the
following peptide mapping/sequencing were already disussed. I would like
to add one comment from the SDS-PAGE side. C-B stained PVDF blot will show
a (very) faint band if you have about 0.05 - 0.1 ug of protein. Less if
the protein came from a mini gel. This translates to about 1-2 picomoles
of 50 kD protein on the blot. Even with all the technical progress for in
situ digestions it may be difficult to obtain anything from this quantity
(on PVDF). It might be possible if you do in gel tryptic digest followed
by MS analysis, but even that does not look like a standard, easy to
perform protocol. If the protein is not N-terminally blocked - you might
be able to sequence it depending on the protein sequencer you have. But if
the protein is partially blocked and only 0.25-.5 picomoles are
available...
Jacek Mozdzanowski
Bioanalytical Sciences
SmithKline Beecham