DTNB (5,5'-Dithio-bis (2-nitrobenzoic acid) also known as Ellman’s reagent for determination of total sulfhydryl groups, protein-bound sulfhydryl groups, and free sulfhydryl groups in biological samples.

1. BARKATULLAH UNIVERSITY
Department of Biochemistry & Genetics
Paper code: E1 304: PROTEOMICS & GENOMICS
Unit: IV
Topic: DTNB ASSAY
BY
Ms. NIDHI PURANIK , PhD
Faculty Member
Department of Biochemistry & Genetics

2. DTNB (5,5'-Dithio-bis (2-nitrobenzoic acid)
(Ellman’s reagent)
APPLICATION
1. Quantitative Determination of Peptides by Sulfhydryl (-SH) Groups.
2. Determination of total sulfhydryl groups, protein-bound sulfhydryl groups,
and free sulfhydryl groups in biological samples using DTNB

3. • Ellman’s reagent 5,5′ -dithiobis (2-nitrobenzoic acid) (DTNB) was first introduced in
1959 for the estimation of free thiol groups
• The procedure is based on the reaction of the thiol with DTNB to give the mixed disulfide
and 2-nitro-5-thiobenzoic acid (TNB) which is quantified by the absorbance of the anion
(TNB2-) at 412 nm.
• The reagent has been widely used for the quantitation of thiols in peptides and proteins. It
has also been used to assay disulfides present after blocking any free thiols (e.g., by
carboxymethylation) and reducing the disulfides prior to reaction with the reagent.
• It is also commonly used to check the efficiency of conjugation of sulf-hydryl-containing
peptides to carrier proteins (such as maleimide activated keyhole limpet haemocyanin,
KLH) in the production of antibodies and to ensure that cysteine labeled sythetic peptides
are coupled to affinity resins.

4. Materials
1. Reaction buffer: 0.1 M phosphate buffer, pH 8.0.
2. Denaturing buffer: 6 M guanidinium chloride, 0.1 M Na2HPO4, pH 8.0
3. Ellman’s solution: 10 mM (4 mg/mL) DTNB in 0.1 M phosphate buffer,
pH 8.0.
4. Dithiothreitol (DTT) (Boerhinger or Calbiochem) solution: 200 mM in
distilled water

5. Analysis of Free Thiols
1. It may be necessary to expose thiol groups, which may be buried in the interior of the
protein. The sample may therefore be dissolved in reaction buffer or denaturing buffer.
A solution of known concentration should be prepared with a reference mixture
without protein. Sufficient protein should be used to ensure at least one thiol per
protein molecule can be detected; in practice, at least 2 nmol of protein (in 100 μL)
are usually required.
2. Sample and reference cuvets containing 3 mL of the reaction buffer or denaturing
buffer should be prepared and should be read at 412 nm. The absorbance should be
adjusted to zero (Abuffer).
3. Add 100 μL of buffer to the reference cuvet.

6. 4. Add 100 μL of Ellman’s solution to the sample cuvet. Record the absorbance
(ADTNB).
5. Add 100 μL of protein solution to the reference cuvet.
6. Finally, add 100 μL protein solution to the sample cuvet, and after thorough mixing,
record the absorbance until there is no further increase. This may take a few minutes.
Record the final reading.
7. The concentration of thiols present may be calculated from the molar absorbance of
the TNB anion.

7. Analysis of Disulfide Thiols
1. Sample should be carboxymethylated or pyridethylated without prior reduction. This
will derivatize any free thiols in the sample, but will leave intact any disulfide bonds.
2. The sample (at least 2 nmol of protein in 100 μL, is usually required) should be
dissolved in 6 M guanidinium HCl, 0.1 M Tris-HCl, pH 8.0 or denaturing buffer,
under a nitrogen atmosphere.
3. Add freshly prepared DTT solution to give a final concentration of 10–100 mM. Carry
out reduction for 1–2 h at room temperature.
4. Remove sample from excess DTT by dialysis for a few hours each time, with two
changes of a few hundred mL of the reaction buffer or denaturing buffer.
Alternatively, gel filtration into the same buffer may be carried out.
5. Analysis of newly exposed disulfide thiols can thus be carried out as previous.