Radioiodination of Cellular Proteins

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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[¹²⁵ I] (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[¹²⁵ I] (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[¹²⁵ I] (∼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

Figure 7.10.1 Nitrogen cavitation device. 1, High‐pressure vessel (nitrogen bomb); 2, nitrogen filling tube; 3, valves regulating the flow of nitrogen to and from the vessel; 4, pressure gauge; 5, discharge tube.

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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. 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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Radioiodination of Cellular Proteins

Abstract

Table of Contents

Materials

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

Basic Protocol 2: Radioiodination of Membrane‐Solubilized Proteins

Support Protocol 1: Membrane Preparation by Homogenization

Support Protocol 2: Membrane Preparation by Nitrogen Cavitation

Basic Protocol 3: Lactoperoxidase‐Catalyzed Radioiodination of Soluble Proteins

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

Figures

Videos

Literature Cited