At 10:56 AM 6/12/00 +0100, mike.naldrett wrote:
>Does anybody have experience of endo Glu-C digestions in ammonium bicarbonate
>pH 8.0 cleaving between SER and GLY residues? More completely the
sequence is
>YLRSGGKP. Any clues would be welcome.
>
Mike-
I've never experienced that, but in the Allen (Laboratory Techniques in
Biochemistry &Molecular Biol v9, Elsevier, 1989) he says at the top of
page 91 that cleavage at Ser has been seen in some proteins. I was hoping
that he would say that it has been seen at phospho-Ser, because I seem to
recall someone having noted that, but I'm not sure if this is even a
correct memory on my part. Does anyone else recall/have a ref to that?
The presence of basic residues in the closely flanking segments makes me
think of that possibility of phosphorylation, though.
Does this cleavage seem to be near-quantitative? If the enzyme were
cleaving at phospho-Ser, the basic buffer conditions ought to promote
dephosphorylation which might take place fast enough to limit the amount of
phosphorylated sites the enzyme had a chance at. If you have more sample
for another shot, pre-incubation without Glu-C for some time ought to
further depress the amount of this cleavage seen later.
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
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