I am attempting to label engineered thiols in a viral
protein using various mercury compounds. These include
methyl mercury(II), PCMBS, and Tetrakis. Mercury addition
is quantitated by ICP-MS. My problem is that I only get
limited modification, maximum about 50%. Steric hindrance
should not be a problem, going by the published crystal
structure.
My question concerns the reduction step. I reduce with
10-25 mM DTT and then gel filtrate quickly on a PD-10.
What are the products of DTT reduction?
Are they mixed disulfides at the Cys residue?
Could these be limiting my Hg reaction?
Is TCEP (Tris[carboxyethylphosphine]hydrochloride a
better reductant for this purpose?
thanks
Norman Watts
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Norman Watts, PhD.
Laboratory of Structural Biology
NIAMS, National Institutes of Health
Bethesda, MD 20892-2717
U.S.A.
E-mail: watts@calvin.niams.nih.gov
Phone: 301-402-3418
Fax: 301-480-7629
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