Norman
You have a great excess of DTT, so the protein should have free cysteines.
However, any contaminating zinc ions or other metallic ions may protect
the protein SH groups. Moreover, any DTT surviving your "quick" PD-10
separation will mop up the mercuric ions. It is also possible that you
must denature the protein to fully reduce it!
Why not use TCEP as reductant? You may be able to avoid the PD-10. You may
need to include a chelating agent that does NOT bind Hg. If you do need
to use the PD-10, strip the metals from it with a good chelating agent (eg
dithizone or o-phenanthroline or EDTA).
Incidentally, I assume that you are aware that Methyl mercury is EXTREMELY
DANGEROUS. A few years ago a scientist last her life by making contact
with a vanishingly small amount. Do be careful if you MUST use it.
Personally, I wouldn't go near it!
Ken
Kenneth A. Walsh
Emeritus Professor of Biochemistry
Box # 357350
University of Washington
Seattle WA 98195
walsh@u.washington.edu
Phone 206-543-1768
FAX 206-685-9231
On Thu, 17 Feb 2000, Norman Watts wrote:
> 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
>
> ------------------------------------------------------------
>
> 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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