Randy,
There are HUNDREDS of variations (mostly minor) on the use of Ellman's
reagent for SH analysis. In general, use a slightly alkaline reaction pH
e.g., pH 8; at least a 10-fold molar excess of Ellman's reagent to
theoretical SH concentration; an extinction coefficient ranging from
~12,500 to 14,500 depending on additional additives present in the reaction
buffer, e.g., EDTA, denaturants like GdmCl, Urea, or SDS; and cysteine to
prepare a standard curve. Some researchers like to use glutathione instead
of cysteine because the thiol group in the former is in a more
"peptide-like" environment.
A real protocol for SH peptide analysis can be found on p 116 of SPPS (2nd
edition, 1984) by Stewart and Young. Contact me if you want additioonal
details.
Regards,
Dan L. Crimmins
Washington University School of Medicine
Dept. Pathology/Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone: 314-454-8514; Fax: 314-454-5208
e-mail: crimmins@labmed.wustl.edu