Date: Mon, 20 Jan 1997 23:08:29 GMT
Message-Id: <v02140b04af093bb33d67@[134.197.50.216]>
From: schooley@unr.edu (David A. Schooley)
Subject: Another Question on Reduction by TCEP
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Dear all,
While you are answering Peter's question about the mechanism of
TCEP reduction, I would like to know if anyone knows a good pmol scale
protocol for COMPLETE reduction of disulfides with TCEP? Bill Grey's
elegant work on TCEP all deals with deliberate partial reductions for
determining disulfide connectivity. Almost a year ago I asked a new
graduate student to try to work out a complete reduction/alkylation with
TCEP using ~10 pmol BSA as a model. He couldn't get complete reduction,
despite changing all sorts of conditions, but did get partial reductions.
When I began to suspect that the problems were not with the TCEP
but with the new grad student, I asked him to see what he could do with pH
8.5, 6 M guanidine HCl, excess DTT, standard stuff. Immediately he got
high recoveries of fully reduced and alkylated BSA. I gained new respect
for the grad student and lost interest in TCEP.
David
David A. Schooley
Dept. of Biochemistry/330
Univ. of Nevada
Reno, NV 89557
schooley@med.unr.edu
tel: (702) 784-4136; fax (702) 784-1419