Preparation of an Iodinated Radioligand

C. Diane True1

1 NEN Life Science Products, North Billerica, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Appendix 3A
DOI:  10.1002/0471142301.nsa03as1
Online Posting Date:  May, 2001
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Abstract

Radioactive tracers are useful for receptor measurement and for radioimmunoassays because of the ease with which radioactive ligands are detected. Typical receptor measurement methods include whole tissue receptor assays, autoradiography, and cell and membrane binding assays. The isotopes most commonly used in receptor measurement studies are tritium, carbon‐14, and iodine‐125, with the choice of isotope most often based on the specific activity required and the sensitivity and type of detection system available. Its high specific activity and easy detection make I‐125 ideal for labeling large peptides and proteins. While the short half‐life (60 days) of iodine requires frequent replacement with freshly iodinated compounds, this is also an advantage, as unused portions of radioligand can be held for decay and disposed of as nonradioactive waste. This unit describes the iodination of proteins or peptides using the 125I‐labeled Bolton Hunter reagent, a convenient, easy‐to‐use, nonoxidizing labeling reagent.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • [125I]Bolton‐Hunter reagent ([125I]BH, NEX‐120) in a Nensure vial with charcoal trap (NEN)
  • Peptide or protein to be labeled
  • 0.05 M sodium phosphate buffer, pH 7.2 to 7.5 ( appendix 2A), or 0.1 M borate buffer, pH 8.0 to 8.5 (see recipe)
  • 0.9% NaCl
  • 0.2 M glycine (see recipe)
  • Sephadex G‐25 prepared columns (5 cm tall, 9.1‐ml bed volume; e.g., PD‐10 columns, Pharmacia Biotech)
  • Gelatin eluting buffer (see recipe)
  • Dilution buffer (see recipe)
  • Instant thin‐layer chromatography (ITLC) paper (Gelman)
  • Chromatography development chamber
  • 12 × 75–mm test tubes
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Figures

Videos

Literature Cited

Literature Cited
   Bolander, F.F. Jr. and Fellows, R.E. 1975. Growth hormone covalently bound to sepharose or glass: Analysis of ligand release rates and characterization of soluble radiolabeled products. Biochemistry 14:,2938‐2942.
   Bolton, A.E. and Hunter, W.M. 1973. The labelling of proteins to high specific radioactivities by conjugation to a 125I‐containing acylating agent. Biochem J. 133:529‐539.
   Bolton, A.E., Bennie, J.G., and Hunter, W.M. 1976. Innovations in labeling techniques for radioimmunoassays. In Proceedings of the 24th Colloquium Brugge, pp. 687‐693.
   Coutts, S.M. and Reid, D.M. 1978. Purification of small peptides labeled with Bolton‐Hunter reagent. Anal. Biochem. 91:446‐450.
   Langone, J.J. 1989. Radioiodination by use of the Bolton‐Hunter and related reagents. Methods Enzymol. 70:221‐239.
   Silver, D. 1988. Radioiodination techniques. In Isotopes: Essential Chemistry and Applications II (J.R. Jones, ed.) pp. 40‐55. Royal Society of Chemistry, London.
Key Reference
   Bolton and Hunter, 1973. See above.
  The original paper using [125I]Bolton‐Hunter reagent to label peptides and proteins.
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