The best enzyme other than trypsin for us has been endoproteinase lys-c,
which works equally well in membrane, gel and solution digestions.
Clostripain (ARG-c from Promega) has worked equally well for us on PVDF
and solution digestions but we have no in-gel experience on this
enzyme. The Boeringer-Mannhein version has always been difficult for us
but it has been quite a few years since we tried them again. We have found
the digestion buffer needs CaCl2 (2 mM) and DTT (4-5 mM) to work.
Endoproteinase Asp-N has been successful for PVDF and solution digestions
but again no experience with in-gels. Buffer conditions were same as with
trypsin.
Glu-c, V8 protease, ASP/GLU-C protein or whatever we choose to call this
enzyme has produced good results in our hands although there has been
occasional problems with poor peptide maps. As Gary earlier mentioned, we
definitely see ASP cleavages at pH 7.8 almost everytime we have used the
enzyme.
A poster at the past ABRF meeting (P4 by amino Woods) showed data
explaining this ASP/GLU cleavage problem. Apparently when an
enzyme:substrate ratio such as 1:5 through 1:50 was used, there was
definitely ASP clips in the test sample. Higher enzyme:substrate ratio's
such as 1:500 produced no ASP clips, only GLU clips. I am not sure but I
thought the original work on this protease used higher E:S ratio's and the
ratio's of 1:20 or less have become widely used because they are necessary
for success of the in-gel or on-PVDF techniques.
Regarding the low pH glu-c method, I have not tried this. We were getting
pretty nice ASP clips at the higher pH already.
Hope this helps.
Joe Fernandez
Joseph Fernandez
Associate Director
Protein/DNA Technology Center
The Rockefeller University
1230 York Ave.
NY,NY 10021
Phone: 212-327-8869
FAX: 212-327-8620
Email: fernaj@rockefeller.edu
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