About a year ago I did a project which required searching for a specific asp
peptide in a protease digest of a myosin fragment by sequencing peaks collected
from HPLC. The resultant digest yielded more glu peptides than asp peptides as
well as one peptide with an N-terminal Serine (cleaved at an RS bond) the
researcher used the Boehringer enzyme (1420-488) in a 50 mM sodium phosphate ph8
buffer for 6 hour at 37 degrees. The best I can tell, they used a ratio of about
50:1.
The enzyme package insert states that the enzyme cleaves at both N-terminal asp
and cysteine and recommends a 200:1 to 20:1 (wt:wt) ratio for 2-18 hrs at 37
degrees.
I don't know if you can eliminate cleavage at glu by using shorter digestion
time and less enzyme.
Linda
Big Apple Minute: The weather forcasters led us to believe we would wake up to 5
inches of snow this morning. I did not share my daughter's hope that school
would be closed, but was disappointed when there wasn't even enough white stuff
to cover the pavement. The tree and bush branches, heavy laden, afforded some
compensation by their beauty.
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Linda Siconolfi-Baez
Core Protein Sequencing Center
SUNY Health Science Cntr.
450 Clarkson Ave.
Box 8
Brooklyn, N.Y. 11203
718-270-1267 (phone)
718-270-3316 (fax)
lsiconolfi-baez@netmail.hscbklyn.edu
http://www.hscbklyn.edu/SUNY/Biochem/Bioparts/ProtSeq.html
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______________________________ Reply Separator _________________________________
Subject: Asp-N
Author: <dangott@mail.chem.tamu.edu (Lawrence J. Dangott) > at Internet
Date: 2/5/99 1:58 PM
Howdy y'all,
We need to know about Asp-N specificity when used in solution digests of
proteins. We see reports of cleavage after Asp, Glu and Cys (non-alkylated)
and would like to know your comments. Our requirement would be strict
fidelity to Asp. Any comments on suppliers are also welcome.
Thanks for your help.
Larry
********************************************************
Larry Dangott, Ph.D.
Protein Chemistry Laboratory
Department of Chemistry
Texas A&M University
College Station, TX 77842-3012
(409) 845-2965 Office
(409) 845-2433 Lab
(409) 845-8015 (FAX)
dangott@mail.chem.tamu.edu
quorum.tamu.edu/~proteins