DTNB (Ellman's Reagent) (5,5-dithio-bis-(2-nitrobenzoic acid)

Catalog number: 22582

Thermo Scientific™  Related applications: Protein Assays and Analysis

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Thermo Scientific Pierce Ellman's Reagent (DTNB) reacts with sulfhydryl groups to yield a colored product, providing a reliable method to measure reduced cysteines and other free sulfhydryls in solution.

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

Features of Ellman's Reagent:

Sulfhydryl assay—reacts quantitatively with free (reduced) sulfhydryl groups (—SH) to yield detectable TNB product
Colorimetric detection—colored product enables spectrophotometric measurement in cuvettes or microplates (λmax = 412nm; ε = 14,150/M·cm)
Proven method—well-characterized chemistry provides quantitation of peptide or protein sulfhydryl groups using extinction coefficient calculation or by comparison to a cysteine standard

Reagent Properties
• Alternative names: DTNB, Ellman's Reagent
• Chemical name: 5,5'-dithio-bis-(2-nitrobenzoic acid)
• Reactive toward: Sulfhydryl groups (—SH)
• Chemical formula: C14H8N2O8S2
• CAS number: 69-78-3
• Molecular weight: 396.35
• Form: Yellow crystalline powder

DTNB reacts with a free sulfhydryl group to yield a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB). The target of DTNB in this reaction is the conjugate base (R—S-) of a free sulfhydryl group. Therefore, the rate of this reaction is dependent on several factors: (1) the reaction pH, (2) the pKa' of the sulfhydryl and (3) steric and electrostatic effects. TNB is the colored species produced in this reaction and has a high molar extinction coefficient in the visible range. The molar extinction coefficient of TNB was originally reported by Ellman (1959) to be 13,600M-1 cm-1 at 412 nm and pH 8.0. Consequently, this value has been cited frequently in the literature. Later studies have shown, however, that the molar extinction coefficient is more accurately reflected by a value of 14,150M-1 cm-1 at 412 nm. The extinction of TNB is not affected by changes in pH between 7.6 and 8.6. However, the extinction of TNB is different in other solvents.
For Research Use Only. Not for use in diagnostic procedures.


Chemical Reactivity: Thiol
Product Size: 5 g

Contents & storage

Store protected from moisture at 4°C. Shipped at ambient temperature.


Manuals & protocols