Ellman"s Reagent (DTNB) reacts with sulfhydryl groups to yield a colored product, providing a reliable method to measure reduced cysteines and other free sulfhydryls in solution. Ellman"s Reagent (5,5"-dithio-bis-[2-nitrobenzoic acid]) is used to estimate sulfhydryl groups in a sample by comparing to a standard curve of a sulfhydryl-containing compound such as cysteine. Alternatively, sulfhydryl groups can be assayed using the extinction coefficient of TNB (14,150M-1cm-1 at 412nm). Ellman"s Reagent has been used for applications other than sulfhydryl estimation, such as for determining alkylthiols by HPLC using a pre-column derivatization procedure and to study thiols in the active site of several enzymes. Highlights: ?Sulfhydryl assay – reacts quantitatively with free (reduced) sulfhydryl groups (–SH) to yield detectable TNB product ?Colorimetric detection – colored product enables spectrophotometric measurement in cuvettes or microplates (λmax = 412nm; ε = 14,150/M?cm) ?Proven method – well-characterized chemistry provides quantitation of peptide or protein sulfhydryl groups using extinction coefficient calculation or by comparison to a cysteine standard