ORGLIST: Thio-Mercapto Reaction

Date view Thread view Subject view Author view Attachment view

From: Norman Watts (Norman_Watts$##$nih.gov)
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.

n
__________________________________________________

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

_______________________________________________
ORGLIST - Organic Chemistry Mailing List
Website / Archive / FAQ: http://www.orglist.net
To post a message (TO EVERYBODY) send to everybody$##$orglist.net
To unsubscribe, send to everybody-request$##$orglist.net the message: unsubscribe your_orglist_password your_address



Date view Thread view Subject view Author view Attachment view

This archive was generated by hypermail 2.1.4 : Thu Feb 18 2010 - 16:58:10 EST