Perhaps the DP cleavage occurs after you have performed your digest.
Are you stopping the digest with 10%TFA, or running HPLC with 0.1%TFA?
good luck,
Paul
Deb McMillen wrote:
> 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
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