Tryptic digest problems

Brett S. Phinney brettsp at mail.utexas.edu
Tue Jul 30 09:05:31 EST 1996


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. 

Any help would be appreciated

Thanks a lot in advance
-- 
Brett Phinney
Cell Research Institute
University of Texas, Austin



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