tryptic digest

Deb McMillen (mcmillen@morel.uoregon.edu)
Tue, 24 Feb 1998 09:49:01 -0800 (PST)

Hi, all,
I have a client who is getting cleavage of a protein between D and P with
trypsin (Sigma Type 13 TPCK treated bovine pancreatic). When he starts
the cleavage, the protein is intact. Of course, the first thing that
comes to mind is how labile the DP bond is, but doesn't it take at least
acidic conditions perhaps with elevated temperature to break the bond?
The tryptic digestion (partial) is done at room temperature in 10 mM
Hepes, 150 mM NaCl, 0.1 mM EDTA, 0.5mM DTT, 10% glycerol pH 7.8. They are
using 50 micrograms of trypsin per 500 micrograms of protein. Is
one of the reaction mixture components a strong (or weak) enough acid to
attack the DP bond? This morning they are checking the pH of the
reaction mixture--the typsin is reconstituted in water before addition to
the protein in 10mM Hepes (not much buffering capacity) at pH 7.8, but it
has always been 7.8 before.

Thanks for any suggestions, Deb

Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403