Ting,
Trypsin only has endopeptidase activity. Thus, if it first cleaves
between the KK to give you a peptide starting KT, an exopeptidase would be
required to cleave off the N-terminal K.
Re. the non-quantitative cleavage at the other end, since you already have
a healthy enzyme-to-substrate ratio this may mean that NPK does not fill
the P1-4 recognition sites sufficiently to give efficient endopeptidase
cleavage.
Vernon Shoup wrote of finding more complete cleavage at multiple basic
sites with the Promega modified enzyme. If anyone knows that Promega's
modifications specifically alter the endo-only properties, please comment.
Regards,
John
John Hempel, PhD Ph (412) 624 0161
University of Pittsburgh FAX (412) 624 4759
Department of Biological Sciences
Clapp Hall 301
Pittsburgh PA 15260 USA
email: hempel@psc.edu
http://www.pitt.edu/~biology/faculty/hempel.html
At 10:38 AM 8/23/00 -0600, Ting Wong wrote:
>Hi, everybody:
>
>On analysis of a tryptic map, I have the following fragments:
>
>KT..........YKNPK
>KT...........YK
>T.............YKNPK
>T..............YK
>
>I know there is a lysine before the K in the sequence, so this is
>apparently the result of incomplete digestion of the sequence:
>
>KKT............YKNPK
>
>Does anybody have suggestion on how to achieve complete digestion to
>have a clean-cut map? Thanks in advance for suggestions.
>
>Ting Wong
>Biomira Inc.
>
>
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