125I Labeling of Proteins


Iodination of proteins is a common method of adding a tracer with high specific activity to your protein of interest. Several different methods are available, and these differ in terms of the amino acids that become labeled and in the reaction conditions that are used. Use the selection tables below to help you choose the best option. You may also find it convenient to contact our OnPoint custom conjugation service for help with this procedure. One commonly-used iodination method, Bolton-Hunter, is described below in detail.

Iodine isotopes have relatively short half-lives, 60 days for 125I. In addition, iodine-labeled proteins are subject to loss of activity and physical degradation due to the isotopic decay. For these reasons, iodine-labeled proteins generally should be used as soon as possible, generally within 30 days or less after labeling.


Choosing an iodination technique

125I properties


Recommended applications


Decay mode



High efficiency for measurement (autoradiography, gamma counting, scintillation counting)
Most commonly used in RIA and receptor ligand binding assays
Well suited for SPA (Scintillation Proximity Assay)

60.14 days

Electron capture

Ka X-ray: 0.027 MeV (112.5%)
Kb X-ray: 0.031 MeV (25.4%)
Gamma: 0.035 MeV (6.5%)


Iodination techniques

The choice of a labeling technique depends largely on the particular amino acids available for labeling in your protein or peptide, and on its stability to the reaction conditions. PerkinElmer OnPoint custom services can perform any of the iodination techniques below and consult on the best technique for your needs.

Reaction method


Applicable to

Bolton-Hunter Reagent

Custom labeling:
125I: NEX088

Bolton-Hunter reagent is the N-hydroxysuccinimide ester of iodinated p-hydroxyphenylpropionic acid. The active ester acylates terminal amino groups with the iodinated p-hydxyphenylpropionic residue, effectively introducing radioactive iodine into proteins and peptides. A non-oxidative technique, it is less harsh to proteins than alternative methods.

Conjugation of terminal amino groups. Applicable to peptides and proteins containing lysine residues. The mono-iodinated form is generally recommended for most Bolton-Hunter iodinations.

Bolton-Hunter Reagent

Custom labeling:
125I: NEX088

Same as mono-iodinated Bolton-Hunter Reagent

Double the specific activity (or 4400 Ci/mmol) for each molecule of reagent. Use when the extra sensitivity is important relative to decrease in stability.


Custom labeling:
125I: NEX083

Lactoperoxidase catalyzes the oxidation of iodide using hydrogen peroxide as the enzyme substrate. It is a milder oxidative technique than Chloramine-T.

Applicable to peptides and proteins naturally containing tyrosine, or chemically modified to introduce tyrosine (especially applicable if they contain easily oxidized methionine)


Custom labeling:
125I: NEX084

Chloramine-T (p-toluene sulfonochloramine) is an effective method of labeling a variety of proteins and peptides. This oxidative method involves exposure of the substrate to Chloramine-T in the presence of NaI, 125I-, for a short time and produces high specific activity proteins or peptides labeled with carrier-free radioiodine, but can be harsh.

Substitution of 125I into tyrosine residues in oxido-reducing reaction. Applicable to peptides and proteins naturally containing, or chemically modified to introduce either tyrosine or histidine.

Exchange Labeling with Sodium Iodide
Custom labeling:
125I: NEX086

Appropriate leaving groups exchange with 125I in solvents or melts. (A melt is a heated reaction performed in a solid state, without solvent.) Catalysts may improve yields and reliability as well as shorten reaction times.

Exchange aliphatic or aromatic bromide or non-radioactive iodide. Exchange aromatic amines through diazonium salts or stabilized triazenes.


Custom labeling:
125I: NEX244

A solid phase oxidative method similar to the Chloramine-T method. It is generally considered to be milder, because the reaction takes place on the surface of the oxidant, minimizing exposure to the substrate.

Iodogen is a water insoluble oxidizing agent which can react with 125I to form a highly reactive mixed halogen species. This intermediate can add radioactive iodine atoms to tyrosine or histidine side chain rings.

Other Custom Methods


Propose a specific method to PerkinElmer for review and, if feasible, we will use it to perform your radioiodination.


Iodination products and catalog numbers

Selecting the best 125I formulation for your needs.


Iodination citations

General review of iodination

  • Robert H. Seevers and Raymond E. Counsell, Radioiodination techniques for small organic molecules. Chem. Rev. 82, 575-590. (1982) Link


  • Bolton, A. and Hunter, W. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem. J. 133, 529-539 (1973) Link


  • Hunter, V. and Greenwood, F. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194, 495-496 (1962) Link
  • Greenwood, F. et al. The preparation of I-131-labeled human growth hormone of high specific radioactivity. Biochem. 89, 114-123 (1963) Link


  • Marchalonis, J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem.J. 113, 299-305(1969) Link
  • Morrison, M. Lactoperoxidase-catalyzed iodination as a tool for investigation of proteins. Meth. Enzymol. 70, 214-220 (1980) Link


  • Fraker, P. and Speck, J. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem. Biophys. Res. Commun. 80, 849-857 (1978) Link


Other PerkinElmer products for labeling proteins


Custom Iodination

PerkinElmer offers custom 125I radioiodination labelling services. If you are interested in having your peptide or protein iodinated please contact our custom teams:

Custom Services