Protein Production, Purification, and Analysis > Detection and Assay Methods

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

In Vitro Radiolabeling of Peptides and Proteins

Ton N.M. Schumacher1,  Theodore J. Tsomides1

1Massachusetts Institute of Technology, Cambridge, Massachusetts

Publication Name: 
Current Protocols in Protein Science
Unit Number: 
Unit 3.3
DOI: 
10.1002/0471140864.ps0303s00
Online Posting Date: 
May, 2001
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

Abstract

Radiolabeling of peptides or proteins is often performed to enhance the sensitivity of detection, to quantitate the binding of peptides to other molecules, or for radioimmunoassays. This unit presents a variety of assays for radiolabeling peptides and proteins with 125I, 131I, 14C, and 3H.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Iodination at Tyrosine or Histidine Residues Using Iodo-Beads
  • Alternate Protocol 1: Iodination at Tyrosine or Histidine Residues Using Chloramine T or Iodogen
  • Alternate Protocol 2: Iodination at Tyrosine or Histidine Residues Using Lactoperoxidase
  • Support Protocol 1: Equimolar Iodination to Give High Yields of Product
  • Support Protocol 2: Separation of Unlabeled Peptide from Iodinated Products by HPLC
  • Basic Protocol 2: Iodination at Lysine Residues or N-Terminus Using Bolton-Hunter Reagent
  • Basic Protocol 3: 14C or 3H Labeling at Lysine Residues or N-Terminus by Acetylation Using Anhydride
  • Alternate Protocol 3: 14C or 3H Labeling at Lysine Residues or N-Terminus by Reductive Alkylation
  • Alternate Protocol 4: 14C or 3H Labeling at Cysteine Residues Using Iodoacetic Acid or Iodoacetamide
  • Basic Protocol 4: 3H Labeling at Tyrosine Residues by Reduction of 127I-Labeled Peptide
  • Basic Protocol 5: Introduction of Labeled Amino Acid Residues During Peptide Synthesis
  • Alternate Protocol 5: Selective Labeling on N-Terminus During Peptide Synthesis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Iodination at Tyrosine or Histidine Residues Using Iodo-Beads

 Materials
  • Iodo-Beads (Pierce)
  • 0.1 M sodium phosphate, pH 6.0 or pH 8.5
  • Carrier-free sodium iodide-125 (Na125I; Amersham, Du Pont NEN, or ICN Biomedicals) or sodium iodide-131 (Na131I)
  • 0.1 M sodium hydroxide
  • Peptide, ideally dissolved in £0.4 ml of water or 0.1 M sodium phosphate
  • Methanol
  • HPLC solvent A: 0.1% (v/v) trifluoroacetic acid (TFA) in water
  • HPLC solvent B: 0.1% (v/v) TFA in acetonitrile
  • Filter paper
  • 100-µl syringe with beveled tip, or hot pipettor (i.e., dedicated to radioactive use)
  • Luer-tipped syringes (1-, 5-, and 20-ml) and one 23-G needle
  • Solid-phase extraction device (e.g., Sep-Pak or Sep-Pak Plus C18 cartridge, Waters)
  • Polypropylene or other plastic tubes (12 × 75 mm)

Alternate Protocol 1: Iodination at Tyrosine or Histidine Residues Using Chloramine T or Iodogen

 Additional Materials (also see Basic Protocol 1)
  • Anion-exchange resin, e.g., Dowex 1-X8, chloride form, 100 to 200 mesh (Bio-Rad)
  • BSA solution: 1.0 mg/ml BSA in PBS, with 0.02% sodium azide (NaN3)
  • 1.0 mg/ml chloramine T in 0.1 M sodium phosphate, prepared immediately before use
  • Iodogen (Pierce), dichloromethane, and glass tubes or vials (optional alternate to chloramine T)
  • Saturated solution of tyrosine in water (~0.4 mg/ml at 25°C)
  • 2.5 mg/ml sodium metabisulfite (Na2S2O5) in 0.1 M sodium phosphate, prepared immediately before use (optional alternate to tyrosine solution)
  • Pasteur pipet or 1-ml syringe with glass wool plug inserted at bottom
  • Gel-filtration column, e.g., Sephadex G-10 or G-25 (Pharmacia Biotech), or Excellulose Desalting Column (Pierce; optional alternate to anion-exchange column)

Alternate Protocol 2: Iodination at Tyrosine or Histidine Residues Using Lactoperoxidase

 Additional Materials (also see Basic Protocol 1)
  • 0.2 mg/ml lactoperoxidase in PBS
  • 0.01% hydrogen peroxide (H2O2) in PBS (diluted from 30% stock immediately before use)
  • 2 U/ml glucose oxidase in PBS and 0.1 M D-glucose in PBS (optional alternate to H2O2)

Basic Protocol 2: Iodination at Lysine Residues or N-Terminus Using Bolton-Hunter Reagent

 Materials
  • 0.1 M sodium borate, pH 8.5
  • 0.1% (w/v) gelatin in 0.1 M sodium borate, pH 8.5
  • 125I-labeled Bolton-Hunter reagent (Amersham, Du Pont NEN, or ICN Biomedicals); or substitute unlabeled Bolton-Hunter reagent and iodinate later
  • Peptide, ideally ³1 mg/ml in £100 µl of 0.1 M sodium borate, pH 8.5
  • 1 M glycine in 0.1 M sodium borate, pH 8.5
  • Gel-filtration column, e.g., Sephadex G-10
  • Dry nitrogen tank and outlet tubing fitted with needle
  • Polypropylene or other plastic tubes (12 × 75 mm)

Basic Protocol 3: 14C or 3H Labeling at Lysine Residues or N-Terminus by Acetylation Using Anhydride

 Materials
  • Saturated sodium acetate solution
  • Peptide, ideally ³1 mg/ml in £100 µl H2O or sodium acetate
  • [14C]- or [3H]acetic anhydride (Amersham, Du Pont NEN, ICN Biomedicals, or Sigma)
  • Acetic anhydride, unlabeled (optional)

Alternate Protocol 3: 14C or 3H Labeling at Lysine Residues or N-Terminus by Reductive Alkylation

 Materials
  • 0.2 M sodium borate, pH 9.0
  • Peptide, ideally ³1 mg/ml in 0.1 to 1.0 ml of 0.2 M sodium borate, pH 9.0
  • [3H]sodium borohydride (Amersham, Du Pont NEN, or ICN Biomedicals) or [3H]sodium cyanoborohydride (Amersham)
  • 0.01 M NaOH
  • 37% (12.4 M) formaldehyde stock
  • [14C]formaldehyde and unlabeled sodium cyanoborohydride (optional alternative for labeling with 14C instead of 3H)

Alternate Protocol 4: 14C or 3H Labeling at Cysteine Residues Using Iodoacetic Acid or Iodoacetamide

 Materials
  • 0.5 M Tris×Cl/6 M guanidine×HCl/0.002 M disodium EDTA, pH 8.6
  • Peptide, ideally ³1 mg/ml in 0.1 to 1.0 ml 0.5 M Tris×Cl/6 M guanidine×HCl/0.002 M disodium EDTA, pH 8.6
  • Dithiothreitol
  • 1.0 M NaOH
  • [14C]- or [3H]iodoacetic acid or [14C]iodoacetamide (Amersham, Du Pont NEN, or ICN Biomedicals)
  • Iodoacetic acid or iodoacetamide, unlabeled (optional)

Basic Protocol 5: Introduction of Labeled Amino Acid Residues During Peptide Synthesis

 Materials
  • l-Leucine, unlabeled
  • L-[14C]- or l-[3H]leucine, 1 to 5 mCi
  • Triethylamine
  • 2-(t-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON, Aldrich)
  • Dioxane
  • Diethyl ether, anhydrous
  • 1.0 M HCl, 4°C
  • Separatory funnel
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 3.3.1
    Common peptide radiolabeling reactions. (A) Iodination on tyrosine. (B) Iodination on histidine. (C) Bolton-Hunter iodination on lysine and N-terminus. (D) Acetylation on lysine and N-terminus. (E) Reductive alkylation on lysine and N-terminus. (F) Alkylation on cysteine.

    View Image
  • Figure 3.3.2
    Stoichiometric iodination of the eight-residue peptide LSPFPYDL and HPLC separation of the unlabeled starting material (peak 1) from monoiodinated (peak 2) and diiodinated (peak 3) products. Two milligrams of peptide were iodinated with 20 mCi Na125I and 5 µmol Na127I at pH 7.0 using six Iodo-Beads, injected on a Vydac C4 semipreparative column, and eluted with an acetonitrile gradient of 1%/min at a flow rate of 3 ml/min. The specific radioactivities of the three peaks were <105 cpm/µg, 2.1 × 107 cpm/µg, and 4.6 × 107 cpm/µg, respectively.

    View Image

Literature Cited

Literature Cited
    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.
    Chersi, A., Trinca, M.L., and Camera, M. 1988. 14C-labeling of synthetic peptides. J. Immunol. Methods 110:271-273.
    Crestfield, A.M., Moore, S., and Stein, W.H. 1963. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. Biol. Chem. 238:622-627.
    Edelhoch, H. 1962. The properties of thyroglobulin. VIII. The iodination of thyroglobulin. J. Biol. Chem. 237:2778-2787.
    Eisen, H.N. and Keston, A.S. 1949. The immunologic reactivity of bovine serum albumin labeled with trace amounts of radioactive iodine (131I). J. Immunol. 63:71-80.
    Fraenkel-Conrat, H. and Colloms, M. 1967. Reactivity of tobacco mosaic virus and its protein toward acetic anhydride. Biochemistry 6:2740-2745.
    Fraker, P.J. and Speck, J.C., Jr. 1978. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3,6-diphenylglycoluril. Biochem. Biophys. Res. Commun. 80:849-857.
    Gahmberg, C.G. and Andersson, L.C. 1977. Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride. J. Biol. Chem. 252:5888-5894.
    Greenwood, F.C., Hunter, W.M., and Glover, J.S. 1963. The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123.
    Hubbard, A.L. and Cohn, Z.A. 1975. Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells. J. Cell Biol. 64:438-460.
    Hunter, W.M. and Greenwood, F.C. 1962. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194:495-496.
    Jentoft, N. and Dearborn, D.G. 1983. Protein labeling by reductive alkylation. Methods Enzymol. 91:570-579.
    Marchalonis, J.J. 1969. An enzymatic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113:299-305.
    Marchalonis, J.J., Cone, R.E., and Santer, V. 1971. Enzymic iodination: A probe for accessible surface proteins of normal and neoplastic lymphocytes. Biochem. J. 124:921-927.
    Markwell, M.A.K. 1982. A new solid-state reagent to iodinate proteins: Conditions for the efficient labeling of antiserum. Anal. Biochem. 125:427-432.
    Rizzino, A. and Kazakoff, P. 1991. Iodination of peptide growth factors: Platelet-derived growth factor and fibroblast growth factor. Methods Enzymol. 198:467-479.
    Sarin, V.K., Kent, S.B.H., Tam, J.P., and Merrifield, R.B. 1981. Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal. Biochem. 117:147-157.
    Stewart, J.M. and Young, J.D. 1984. Solid Phase Peptide Synthesis. Pierce Chemical Co. Rockville, Ill.
    Tack, B.F. and Wilder, R.L. 1981. Tritiation of proteins to high specific activity: Application to radioimmunoassay. Methods Enzymol. 73:138-147.
    Thean, E.T. 1990. Comparison of specific radioactivities of human -lactalbumin iodinated by three different methods. Anal. Biochem. 188:330-334.
    Tsomides, T.J. and Eisen, H.N. 1993. Stoichiometric labeling of peptides by iodination on tyrosyl or histidyl residues. Anal. Biochem. 210:129-135.
    Wood, F.T., Wu, M.M., and Gerhart, J.C. 1975. The radioactive labeling of proteins with an iodinated amidination reagent. Anal. Biochem. 69:339-349.
    Wolff, J. and Covelli, I. 1969. Factors in the iodination of histidine in proteins. Eur. J. Biochem. 9:371-377.
 Key References
    Means, G.E. and Feeney, R.E. 1971. Chemical Modification of Proteins. Holden-Day, Inc., San Francisco, Calif.

Excellent overview of side chain chemistries, including but not limited to radiolabeling procedures.

    Tsomides and Eisen 1993. See above.

Describes use of Sep-Pak to remove free iodine, HPLC to separate iodinated products, Edman degradation to identify products, and pH dependence of tyrosine vs. histidine iodination using Iodo-Beads.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.