The Bolton and Hunter Method for Radiolabeling Protein

  • Graham S. Bailey
Part of the Springer Protocols Handbooks book series (SPH)


This is an indirect method in which an acylating reagent (N-succinimidyl-3[4-hydroxyphenyl]propionate, the Bolton and Hunter reagent), commercially available in a radioiodinated form, is covalently coupled to the protein to be labeled (1). The [125I] Bolton and Hunter reagent reacts mostly with the side-chain amino groups of lysine residues to produce the labeled protein. It is the method of choice for radiolabeling proteins that lack tyrosine and histidine residues, or where reaction at those residues affects biological activity. It is particularly suitable for proteins that are sensitive to the oxidative procedures employed in other methods (see Chapters 112, 113, and 115).


Sodium Phosphate Sodium Azide Indirect Method Lysine Residue Histidine Residue 
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  1. 1.
    Bolton, A. E. and Hunter, W. M. (1973) The labeling of protiens to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem. J. 133, 529–538.PubMedGoogle Scholar
  2. 2.
    Langone, J. J. (1980) Radioiodination by use of the Bolton-Hunter and related reagents, in Methods in Enzymology, vol. 70 (Van Vunakis, H. and Langone, J. J., eds.), Academic, New York, pp. 221–247.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Graham S. Bailey
    • 1
  1. 1.Department of Biological and Chemical SciencesUniversity of EssexColchesterUK

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