Radioiodination of Cellular Proteins

Steve Caplan1, Michal Baniyash2

1 NICHD, National Institutes of Health, Bethesda, Maryland, 2 Hebrew University‐Hadassah Medical School, Jerusalem, Israel
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.10
DOI:  10.1002/0471143030.cb0710s15
Online Posting Date:  August, 2002
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Abstract

Despite the advances of non‐radioactive methods of protein labeling, radioiodination remains an important tool for studying proteins. This unit discusses several commonly used methods for radioiodination of proteins for various purposes. Included are several simple, reliable protocols for radioiodination of cell surface proteins on live cells, radioiodination of purified, solubilized membrane proteins, and radioiodination of purified soluble proteins.

     
 
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Table of Contents

  • Basic Protocol 1: Cell Surface Labeling with 125I Using Lactoperoxidase
  • Basic Protocol 2: Radioiodination of Membrane‐Solubilized Proteins
  • Membrane Preparation
  • Support Protocol 1: Membrane Preparation by Homogenization
  • Support Protocol 2: Membrane Preparation by Nitrogen Cavitation
  • Radioiodination of Soluble Proteins
  • Basic Protocol 3: Lactoperoxidase‐Catalyzed Radioiodination of Soluble Proteins
  • Alternate Protocol 1: Chloramine‐T‐Mediated Radioiodination of Soluble Proteins
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Cell Surface Labeling with 125I Using Lactoperoxidase

  Materials
  • 0.5–1 × 108 cells in suspension
  • PBS ( appendix 2A), azide free and ice cold
  • Na[125I] (0.1 mCi/µl; NEN Life Science Products, Amersham Pharmacia Biotech, or ICN Biomedicals)
  • Lactoperoxidase enzyme solution (see recipe)
  • 30% (v/v) hydrogen peroxide stock solution
  • Lactoperoxidase buffer (see recipe), ice cold
  • 10 mM NaI in PBS ( appendix 2A), ice cold
  • Fume hood with double row of 4‐cm‐thick lead bricks
  • γ counter and appropriate counting vials

Basic Protocol 2: Radioiodination of Membrane‐Solubilized Proteins

  Materials
  • 1 ml membrane‐solubilized proteins from 2 × 108 cells (see protocol 3 and protocol 4)
  • Lactoperoxidase beads in 50% suspension (Worthington Biochemical)
  • Na[125I] (0.1 mCi/µl; NEN Life Science Products, Amersham Pharmacia Biotech, or ICN Biomedicals)
  • 3% (w/v) dextrose
  • Triton X‐100 lysis buffer (see recipe) with 0.02% (w/v) BSA
  • PD‐10 columns (10‐ml columns prepacked with Sephadex G‐25 resin; Amersham Pharmacia Biotech)
  • γ counter and appropriate counting vials

Support Protocol 1: Membrane Preparation by Homogenization

  Materials
  • Cells grown in culture or from dissected organs.
  • PBS ( appendix 2A), ice cold
  • Dounce buffer (see recipe)
  • 10× phosphatase inhibitors (see recipe), optional
  • Tonicity restoration buffer (see recipe), ice cold
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • Triton X‐100 lysis buffer (see recipe)
  • Phase‐contrast microscope
  • Glass‐glass (Dounce) homogenizer with pestle B, ice cold
  • 10‐ml conical centrifuge tube, prechilled
  • Ultracentrifuge, Beckman TLA100.3 rotor or equivalent, and ultracentrifuge tubes, 4°C
  • Additional reagents and equipment for cell counting (unit 1.1)

Support Protocol 2: Membrane Preparation by Nitrogen Cavitation

  • Nitrogen cavitation device (e.g., Cell Disruption Bomb, Parr Instrument; Fig. )
  • Nitrogen gas tank

Basic Protocol 3: Lactoperoxidase‐Catalyzed Radioiodination of Soluble Proteins

  Materials
  • 10 mg/ml BSA in PBS
  • PBS ( appendix 2A), ice cold
  • 30% (v/v) hydrogen peroxide stock solution
  • 0.025 M sodium phosphate buffer, pH 7.4 ( appendix 2A), ice cold
  • 1 mg/ml protein sample in PBS
  • Na[125I] (∼0.1 mCi/µl; NEN Life Science Products, Amersham Pharmacia Biotech, or ICN Biomedicals)
  • 1.5 mg/ml lactoperoxidase enzyme (Sigma or Calbiochem) in PBS ( appendix 2A), stored in aliquots at −70°C or prepared fresh
  • 15 mM NaI in PBS, prepared fresh and ice cold
  • PD‐10 columns (10‐ml columns prepacked with Sephadex G‐25 resin; Amersham Pharmacia Biotech)
  • γ counter and appropriate counting vials

Alternate Protocol 1: Chloramine‐T‐Mediated Radioiodination of Soluble Proteins

  • 0.5 M sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • Chloramine‐T (Sigma)
  • Sodium metabisulfite (Sigma)
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Figures

Videos

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. Wiley & Sons, London.
   Kinne‐Saffran, E. and Kinne, R.K. 1989. Membrane isolation: Strategy, technique, markers. Methods Enzymol. 172:3‐17.
   Koch, N. and Haustein, D. 1981. Radioiodination of surface proteins and glycoproteins of lymphocytes by immobilized lactoperoxidase. J. Immunol. Methods 41:163‐171.
   Morrison, M. 1980. Lactoperoxidase‐catalyzed radioiodination as a tool for investigation of proteins. Methods Enzymol. 70(A):214‐220.
Key Reference
   Bailey, G.S. 1994. Labeling of peptides and proteins by radioiodination. In Methods in Molecular Biology, Vol. 32 (J.M. Walker, ed.) pp. 441‐448. Humana Press, Totowa, N.J.
  Contains background information and standard protocols for radioiodination by chloramine‐T‐ and lactoperoxidase‐catalyzed radioiodination.
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