Iodination of Soluble and Membrane‐Bound Proteins

L.E. Samelson1

1 National Institute of Child Health and Human Development, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.11
DOI:  10.1002/0471142735.im0811s11
Online Posting Date:  May, 2001
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Abstract

Coupling of radioactive 125I to soluble proteins is a critical step in a number of immunoassays. Chloramine T‐catalyzed iodination of soluble proteins, such as immunoglobulins, is described in this unit. Protocols using lactoperoxidase‐catalyzed iodination of soluble or membrane‐bound proteins are also described.

     
 
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Table of Contents

  • Basic Protocol 1: Chloramine T–Catalyzed Iodination of Soluble Proteins
  • Alternate Protocol 1: Lactoperoxidase‐Catalyzed Iodination of Soluble Proteins
  • Basic Protocol 2: Lactoperoxidase‐Catalyzed Iodination of Membrane Proteins
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Chloramine T–Catalyzed Iodination of Soluble Proteins

  Materials
  • 10 mg/ml carrier protein in PBS (FCS, BSA, or ovalbumin)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • recipe0.5 M sodium phosphate buffer, pH 7.4
  • 1 mg/ml immunoglobulin (or other protein to be iodinated) in PBS
  • Chloramine T
  • Sodium metabisulfite
  • Carrier‐free Na[125I] (∼100 mCi/ml; Du Pont NEN, Amersham, or ICN)
  • 15 mM NaI in PBS
  • Gel filtration column and resin (e.g., PD‐10 column containing Sephadex G‐25; Pharmacia LKB #17‐0851‐01)
  • Geiger counter
  • γ scintillation counter and tubes

Alternate Protocol 1: Lactoperoxidase‐Catalyzed Iodination of Soluble Proteins

  Additional Materials
  • 30% hydrogen peroxide (H 2O 2)
  • recipe0.025 M sodium phosphate buffer, pH 7.4 (diluted 1:20 from 0.5 M sodium phosphate buffer, pH 7.4)
  • 10 µM  lactoperoxidase in PBS (store at −70°C in aliquots or prepare fresh each time from powder; Sigma)

Basic Protocol 2: Lactoperoxidase‐Catalyzed Iodination of Membrane Proteins

  Additional Materials
  • Tissue culture cells or lymphocytes in suspension
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • recipeSupplemented PBS
  • 20 µM lactoperoxidase in PBS (store at −70°C in aliquots)
  • Carrier‐free Na[125I] (∼100 mCi/ml; Du Pont NEN, Amersham, or ICN)
  • 15 mM NaI in PBS
  • 50‐ml centrifuge tube (Falcon #2058 or 2063)
  • Additional reagents and equipment for harvesting cells ( appendix 3A), washing cells and preparing single‐cell suspension (unit 3.1), and counting viable cells by trypan blue exclusion ( appendix 3A)
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Figures

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Literature Cited

Literature Cited
   Hubbard, A.L. and Cohn, Z.A. 1976. Specific labels for cell surfaces. In Biochemical Analysis of Membranes (A.H. Maddy, ed.) pp. 427‐501. John Wiley & Sons, London.
   Langone, J.J. 1980. Radioiodination by use of the Bolton‐Hunter and related reagents. Methods Enzymol. 70:221‐225.
   Markwell, M.A.K. and Fox, C.F. 1978. Surface‐specific iodination of membrane proteins of viruses and eukaryotic cells using 1,3,4,6‐tetrachloro‐3α, 6α‐diphenylglycouril. Biochemistry 17:4807‐4817.
   Morrison, M. 1974. The determination of the exposed proteins on membranes by the use of lactoperoxidase. Methods Enzymol. 32:103‐109.
   Morrison, M. 1980. Lactoperoxidase‐catalyzed iodination as a tool for investigation of proteins. Methods Enzymol. 70:214‐220.
   Ward, G.M. 1984. Labeling of cell membrane proteins. In Membranes, Detergents, and Receptor Solubilization (J.C. Venter and L.C. Harrison, eds.) pp. 109‐118. Wiley‐Liss, New York.
Key References
   Goding, J.W. 1984. Immunoprecipitation and electrophoretic analysis of membrane proteins. In Molecular and Chemical Characterization of Membrane Receptors (J.C. Venter and L.C. Harrison, eds.) pp. 31‐60. Wiley‐Liss, New York.
  Excellent overview of labeling techniques and protocols for immunoprecipitation and electrophoresis.
   Ward, 1984. See above.
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