Thermo Scientific Pierce Ellman Reagent (DTNB) reacts with sulfhydryl groups to form colored products, providing a reliable method to measure Reduced cysteine and other free sulfhydryl groups in solution.

Ellman's reagent (5,5'-dithio-bis-[2-nitrobenzoic acid]) is used by comparison with a standard curve for compounds containing sulfhydryl groups, such as cysteine , estimate the sulfhydryl groups in the sample. Alternatively, sulfhydryl groups can be measured using the extinction coefficient of TNB (14,150M-1 cm-1 at 412nm). Ellman's reagent has been used in applications other than sulfhydryl estimation, such as HPLC alkylthiol determination using a pre-column derivatization procedure and the study of active site thiols in several enzymes.

Features of Ellman's reagent:

•Sulfhydryl Determination—Quantitatively reacts with free (reduced) sulfhydryl groups (—SH) to produce a detectable TNB product
• Colorimetric Detection—Colored products support spectrophotometric measurements in cuvettes or microplates (λmax = 412nm; ε = 14,150/M·cm)
• Proven analytical methods—Well-characterized chemical reagents enable quantitative determination of peptide or protein sulfhydryl groups using extinction coefficient calculations or comparison to cysteine standards

Reagent properties
• Alias: DTNB, Ellman's reagent
• Chemical name: 5,5'-dithio-bis-(2-nitrobenzoic acid)
• Can be used with the following Group reaction: thiol group (—SH)
• Chemical formula: C₁₄H₈N₂O₈ S₂
• CAS number: 69-78-3
• Molecular weight: 396.35
• Form: yellow crystalline powder

DTCNB and free The thiol groups react to form mixed disulfides and 2-nitro-5-thiobenzoic acid (TNB). In this reaction, the target of DTNB is a coupling group of a free sulfhydryl group (R—S-). Therefore, the rate of this reaction depends on several factors: (1) reaction pH, (2) pKa' of the thiol group, and (3) steric and electrostatic effects. TNB is a colored substance produced by this reaction and has a high molar extinction coefficient in the visible range. Ellman (1959) originally reported the molar extinction coefficient of TNB as 13,600M-1 cm-1 (412 nm, pH 8.0). Therefore, this value is often cited in the literature. However, later studies showed that a value of 14,150M-1 cm-1 at 412 nm more accurately reflects the molar extinction coefficient. The extinction value of TNB is not affected by changes in pH between 7.6 and 8.6. However, the extinction values of TNB differ in other solvents.