Ellman’s assay for in-solution quantification of sulfhydryl groups

Rucha Bhat, Gregory Grover, PhD Contraline Inc., Charlottesville, VA 11/2018

Thiol chemistry is a rapidly expanding field in basic and applied bioscience. As a consequence, the quantitative measurement of -SH groups is a routine task in many applied disciplines where a quick and easy method is much preferred. Electrochemical and fluorimetric assays are very sensitive and accurate but they involve lengthy procedures (complete proteolysis, electrolysis, HPLC separation).

Although spectrophotometric thiol assays such as Ellman’s are less sensitive in comparison, they are rapid and simple and are therefore more popular for the quantification of thiols. Ellman’s reagent is 5,5’-dithio-bis-(2-nitrobenzoic acid), also known as DTNB, a compound used for quantitating free thiol groups in solution. A solution of this compound produces a yellow colored product when it reacts with sulfhydryl groups. This yellow-colored product can be quantified using the SPECTROstar Nano absorbance microplate reader based on its strong absorbance at 412 nm.

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