Ellman’s Reagent INTRODUCTIONEllman’s reagent, 5, 5’-dithio-bis-(2-nitrobenzoic acid), also known as DTNB, is a versatile water-soluble compound for quantitating free sulfhydryl groups in solution. DTNB reacts with a free sulfhydryl group to yield a mixed disulfide and 2-nitro-5-thiobenzoic acid (NTB), a measurable, yellow-colored product with a molar extinction coefficient of 14.15 mM-1 cm-1at 412 nm.DTNB is very useful as a sulfhydryl assay reagent because of its specificity for -SH groups at neutral pH, high molar extinction coefficient, and short reaction time.Chemical Name: 5, 5′-Dithio-bis-(2-nitrobenzoic acid)Molecular Formula: C14H8N2O8S2Molecular Weight: 396.3CAS#: 69-78-3ITEM(S) SUPPLIED Cat. #: BZ355PROTOCOL1. Make 10mM DTNB stock solution by dissolving 40mg DTNB in 10ml DMSO. The stock solution can be stored at 4°C for 3 months. Dilute the stock solution 100-fold with 0.1M Tris-HCl pH 7.5 to make 0.1 mM DTNB working solution.2. Aliquot 950μl of 0.1mM DTNB work solution to each 1.5ml centrifuge tube. Add 50μl test sample and mix by brief vortexing. Set a blank by adding 50μl of 0.1M Tris-HCl pH 7.5 to 950μl of 0.1mM DTNB work solution. Note: The test sample may need to be diluted before applying to the assay and the dilution factor should be recorded. The 50µl test sample applied to the assay reaction should have a sulfhydryl concentration of less than 0.5mM. Concentrations exceeding 0.5mM free sulfhydryl will result in high absorbance values and a less accurate estimation of the concentration based on the extinction coefficient.3. Incubate for 2 minutes at room temperature.4. Measure the absorbance of the test sample with a spectrophotometer against a blank at 412nm.5. Calculate the concentration of free sulfhydryls in the sample from the molar extinction coefficient of NTB (14.15 mM-1 cm-1) as follow:mM free sulfhydryls = Absorbance / (path length x 14.15) x 20 x dilution factorNote: Path length is the cuvette path length in centimeters (cm)20 is the dilution factor of 50µl sample to 1ml assay volumeTransport and storage at room temperature, valid for 12 monthsSpecification: 100mL | 200mL | 500 mL | 1000 mL