Hello all;
I recently performed a sequence analysis on a blotted protein and obtained
a residue with a non standard retention time almost identical with that of
DMPTU. A database search on the first fifteen residues found a perfect
match with a cysteine in the position in question. I found out from the
researcher that the sample had not been alkylated prior to isolation. The
acrylamide derivative of cysteine (PTH-Cys-S-Pam) elutes in this region and
it seemed reasonable to assume that it had been modified because the gel
was not polymerized long enough. However, he has repeated the procedure
three more times with ample polymerization time, but I still see the same
results. Has anyone out there had a similar experience?
Also, if there were enough free acrylamide around to completely derivatize
the cysteine (the peak was the same height as the other residues),
shouldn't it have blocked the N-terminal of the protein as well? I got an
excellent initial yield (~20 pmole) from a single minigel band.
Thanks in advance for any input...
Sandy
Sandy Kielland
Victoria Protein Microchemistry Centre
Department of Biochemistry and Microbiology
University of Victoria
Victoria B.C. CANADA
phone: (250) 7218884
FAX (250) 7218855
Email Kielland@UVic.ca
Webpage http://www.proteincentre.com
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