Estimation of Disulfide Bonds Using Ellman’s Reagent

  • Alastair Aitken
  • Michèle Learmonth
Part of the Springer Protocols Handbooks book series (SPH)


Ellman’s reagent 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) was first introduced in 1959 for the estimation of free thiol groups (1). 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.


Disulfide Bond Reaction Buffer Thiol Group Free Thiol Disulfide Thiol 
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  1. 1.
    Ellman, G. L. (1959) Tissue sulfhydryl groups,. Arch. Biochem. Biophys. 82, 70–77.PubMedCrossRefGoogle Scholar
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    Zahler, W. L. and Cleland, W. W. (1968) A specific and sensitive assay for disulfides. J. Biol. Chem. 243, 716–719.PubMedGoogle Scholar
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    Anderson, W. L. and Wetlaufer, D. B. (1975) A new method for disulfide analysis of peptides. Analyt. Biochem. 67, 493–502.PubMedCrossRefGoogle Scholar
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    Riddles P. W., Blakeley, R. L., and Zerner, B. (1983) Reassessment of Ellman’s reagent. Meth. Enzymol. 91, 49–60.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Alastair Aitken
    • 1
  • Michèle Learmonth
    • 2
  1. 1.Division of Biomedical and Clinical Laboratory Sciences, Membrane Biology GroupUniversity of EdinburghScotland, UK
  2. 2.Department of Biomedical SciencesUniversity of EdinburghScotland

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