In article <01bb7e20$2afd09a0$5d715380 at UT.cc.utexas.edu>, "Brett S.
Phinney" <brettsp at mail.utexas.edu> wrote:
> I was wondering if anybody had any experience doing tryptic digests using
> SDS-PAGE and reversed phase HPLC to separate their protein fragments. I'm
> trying to digest 2 proteins, and collect and sequence the fragments, the
> problem is that when I digest the protein and run it on an SDS-PAGE gel I
> see what I consider is a good digestion if not complete, i.e. the two
> places where the proteins should be are now gone. But when I run the same
> sample on an HPLC using a 2 mm C-18 column, I see 1 big peek and a few
> smaller ones. The big peek is about 50-100 times bigger then the smaller
> ones. Could it be possible that all of my fragments are co-eluting, or I'm
> not getting a good digestion. If I'm not getting a good digestion, then why
> do I not see any of my original protein, or at least very large fragments,
> when I run them on a gel. I should get about 30 fragments after a trypsin
> digestion.
You do not say if you reduced and alkylated the protein prior to digestion
or if you are running reduced SDS gels. If the tryptic digest was done on
native protein then the disulfide bonds would remain intact during HPLC
giving rise to fewer and larger peptides than you would see on a reducing
SDS gel. I would suggest reducton and alkylation prior to digestion or
following digstion but prior to HPLC depending on what your objective is.
Tim Edmunds
