ORGLIST: Thio-Mercapto Reaction

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From: Norman Watts (Norman_Watts$##$
Date: Thu Oct 20 2005 - 10:39:51 EDT

Thank you to those who responded. For reasons not yet clear to me this
seemingly simple problem has proven more difficult than I ever
expected. On the one hand I've struggled to maximize occupancy and on
the other to minimize non-specific labeling. Without tiring you with
the details let me just say that I have done many, many experiments
trying to fine tune this. I have employed a series of mutant proteins
(shifting the Cys residue stepwise from one position to the next) and
non-Cys versions to control for background labeling. I've tried
different buffers and pH conditions, and different reductants (DTT and
TCEP [both soluble and immobilized]. I've tried various labels [PCMBS
and methylmercury (yes I know the hazards), etc, and now the
phenylmercury derivative]. I've monitored the available thiols with
DTNB and the degree of labeling by ICP-MS. I've tried EDTA and
ergothioneine to control metals, and DMSO and immidazole to help
control non-specific binding. Its been a bear, but I'll get it right.
Anyway, thanks for the input.


Norman Watts, Ph. D.
National Institutes of Health
50 South Drive, Rm. 1509
Bethesda, MD 20892-8025
Phone: (301) 402-3418
Fax: (301) 480-7629

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