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

Catalog No. PI22582
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PI22582 5 g
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Catalog No. PI22582 Supplier Thermo Scientific™ Supplier No. LSG22582

Description

Measure peptide cysteines or other free, reduced sulfhydryls (-SH groups) in solution using this thiol-reactive, colored compound (Amax 412nm).

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

A solution of this compound produces a measurable yellow-colored product when it reacts with sulfhydryls. Consequently, Ellman's Reagent 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. Sulfhydryl groups may be estimated in a sample by comparison to a standard curve composed of known concentrations of a sulfhydryl-containing compound such as cysteine. Alternatively, sulfhydryl groups may be quantitated by reference to the extinction coefficient of TNB.

Highlights:

  • Sulfhydryl detection– reacts quantitatively with free (reduced) sulfhydryl groups

  • Colorimetric–yields colored product to enable spectrophotemetric measurement in tubes or microplates

  • Proven assay method– well-characterized chemistry enables quantitation of peptide or protein sulfhydryl groups using extinction coefficient calculation or by comparison to a cysteine standard

Specifications

Product Type DTNB
Quantity 5 g

Frequently Asked Questions (FAQs)

Are there other uses for Thermo Scientific Ellman's Reagent besides estimation of sulfhydryls?

Ellman's Reagent may be used for applications other than the estimation of sulfhydryls in solution. It has been used for the determination of alkylthiols by HPLC using a pre-column derivatization procedure and to study thiols in the active site of several enzymes including thiolase, fatty acid synthase and mevalonate 5-diphosphate decarboxylase.
What should I do if the sulfhydryl content in my sample is lower than expected?

It is possible that the sulfhydryls in the sample are not in reduced (free) state; i.e., have become oxidized. Ensure reduction of sulfhydryls using a reducing agent, then minimize the time delay between assay of the sample and its use in applications that depend on its free sulfhydryl content. Also maintain 1-5 mM EDTA in the sample to chelate divalent metal ions, which can oxidize sulfhydryls.

What are the Quality Specifications for each lot of Thermo Scientific Ellman's Reagent?

The Certificate of Analysis lists if the infrared spectrum conforms to the structure and if the purity ≥ 98%.
How stable are working solutions of Thermo Scientific Ellman's Reagent?

The Ellman's Reagent Solution can be prepared in advance in the recommended reaction buffer (0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA), and will be stable for about 1 week . An Ellman's Reagent Solution prepared in Tris pH 8.0 will also be stable for 1 week. The Ellman's Reagent Solution preapred in PBS, pH 7.2 is only stable for 1-2 days. This chemical is incompatible with strong oxidizing agents, strong bases and is sensitive to the presence of thiols. We have not tested Ellman's Reagent with DMSO.
What is the recommended storage condition for Thermo Scientific Ellman's Reagent?

We recommend storing the product protected from moisture at 4 degrees C. It is susceptible to some hydrolysis by moisture. The product is shipped at ambient temperature.
What are the chemical properties of Thermo Scientific Ellman's Reagent or DTNB?

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
What are the features of Thermo Scientific Ellman's Reagent (Cat. No. 22582)?

Thermo Scientific Ellman's Reagent is provided as 5 grams lyophilized powder, and is pale yellow color. The Sulfhydryl Assay reacts quantitatively with free (reduced) sulfhydryl groups (-SH) to yield detectable TNB product. For colorimetric detection, the colored product enables spectrophotometric measurement in cuvettes or microplates (?max = 412 nm; ? = 14,150/M·cm). Use of this reagent is a proven method, a well-characterized chemistry that provides quantitation of peptide or protein sulfhydryl groups using extinction coefficient calculation or by comparison to a cysteine standard.
What other materials are required for the sulfhydryl group estimation assay that uses Ellman's Reagent?

Here are the required materials:

-Reaction Buffer: 0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA
-Cysteine Hydrochloride Monohydrate, M.W. = 175.6: Thermo Scientific Cat. No. 44889
-Ellman's Reagent Solution: Dissolve 4 mg Ellman's Reagent in 1 mL of Reaction Buffer
-Spectrophotometer to read at 412 nm
-Cuvettes and test tubes for 3 mL volume
Can you summarize the sulfhydryl quantitation protocol for Thermo Scientific Ellman's Reagent?

Sulfhydryl groups may be estimated in a sample by comparison to a standard curve composed of known concentrations of a sulfhydryl-containing compound such as cysteine, preferrably at a working range of 0.1 - 1.0 mM. A volume of 250 µl is required for the sample and standard, mixed with Ellman's Reagent Solution and Reaction Buffer, then incubated for 15 minutes at room temperature. Absorbance is read at 412 nm.

Explain the chemistry of the Thermo Scientific Ellman's Reagent assay?

In 1959, Ellman introduced 5,5'-dithio-bis-(2-nitrobenzoic acid), also known as DTNB, as a versatile water-soluble compound for quantitating free sulfhydryl groups in solution. A solution of this compound produces a measurable yellow-colored product when it reacts with sulfhydryls. Consequently, Ellman's Reagent became 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. 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 to be 13,600M-1 cm-1 at 412 nm and pH 8.0. 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.
When would I use Thermo Scientific Ellman's Reagent?

Thermo Scientific 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.

For Research Use Only. Not for use in diagnostic procedures.