DTNB

DTNB (Ellman’s Reagent) is a chemical used to quantify the number or concentration of thiol groups^[1]. In Vitro:DTNB reacts with the free sulfhydryl side chain of cysteine to form an S-S bond between the protein and a thionitrobenzoic acid (TNB) residue^[1].
The main advantage of DTNB over alternative reagents (e.g., N-ethylmaleimide or iodoacetamide) is in the selectivity of this reagent and in the ability to follow the course of the reaction spectrophotometrically^[1].
1. Preparation of solution
1.1 Reaction Buffer: 0.1M sodium phosphate, pH 8.0, containing 1mM EDTA
2.1 DTNB Solution: Dissolve 4 mg DTNB in 1 mL of Reaction Buffer.

2. Detection
2.1 Add 250 µL of sample to a test tube containing 50 µL of DTNB solution.
Note: For unknown samples, dilution can be performed to ensure that the thiol concentration in the sample is less than 1.0 mM.
2.2 Mix and incubate at room temperature for 15 minutes.
2.3 With a spectrophotometer set to 412 nm and then measure absorbance of each sample.
2.4 Calculate the amount and concentration of sulfhydryls in the sample from the molar extinction coefficient of TNB (14.15 mM^-1 cm^-1).