Ellmans Assay for the Determination of Sulfhydryls

Ellman s reagent has been used not only for the determination of sulfhydryls in proteins and other molecules, but also as a precolumn derivatization reagent for the separation of thiol compounds by HPLC (Kuwata et al., 1982), in the study of thiol-dependent enzymes (Masamune etal., 1989 Tsukamoto andWakil, 1988 Alvear et ai, 1989), and to create sulfhydryl-reactive chromatography supports for the coupling of affinity ligands (Jayabaskaran etal., 1987). Another important use of the compound 

Depending on the conditions, an Ellman s assay can detect as little as 10 nM cysteine concentration. The linearity can extend into the mM range, making the test extremely flexible for different sample situations. 

Dissolve Ellman s reagent (Pierce) in 0.1 M sodium phosphate, pH 8, at a concentration of 4 mg/ml. 

Prepare a set of standards by dissolving cysteine in 0.1M sodium phosphate, pH 8, at an initial concentration of 2 mM (3.5 mg/ml) and serially diluting this solution (1 1) with reaction buffer down to at least 0.125 mM. This will produce five solutions of cysteine for generating a standard curve. If a more dilute concentration range is required, continue to dilute serially until a set of standards in the desired range is obtained. 

Label a set of test tubes according to the standards and samples to be used. Add 250 xl of each standard and sample to the appropriate tubes. If the samples are in a buffer that may significantly change the pH of the reaction buffer, the samples should be buffer-exchanged or dialyzed into 0.1 M sodium phosphate, pH 8, before running the assay. 

Ellman s Reagent Sulfhydryl Disulfide Bond 5-Thio-2-nitrobenzoic 

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