How do we Radiolabel Proteins?

Radiolabeling is an age-old method of labeling proteins using radioactive isotopes. Radiolabeling provides insights not only into the process of protein biosynthesis but also into the turnover of proteins in response to cell cycles or stimuli.

Protein Molecules

Protein Molecules. Image Credit: Design_Cells/

Although there are myriads of other modern protein labeling methods such as biotinylation and fluorescence, radiolabeling remains to be the most reliable and sensitive method of protein detection. One of the main advantages of radiolabeling proteins is that the labeled groups are chemically identical to their naturally occurring analogs, thus enabling the protein to be monitored in their natural environment and without significant loss of material. Radiolabeling of proteins avoids many of the common pitfalls associated with other protein labeling methods including spurious fluorescence, enzymatic activity, or steric problems.

Methods of radiolabeling proteins

Depending on the radionuclide being used there are various radiolabeling strategies available to incorporate a radionuclide into a protein. 1) Direct labeling, 2) indirect labeling via a prosthetic group and, 3) indirect labeling via complexation. The radioactive isotopes can be directly integrated into a protein molecule by electrophilic substitution or indirectly via conjugation. On the other hand, radioactive metals are labeled via complexation with a chelating agent.

Protein radiolabeling has numerous applications in modern-day science. It has helped in our understanding of various biological pathways. For example, the entire insulin signaling pathway was dissected via protein labeling. Proteins can be radiolabeled during the process of translation in the presence of 35S -methionine, or enzymatically by using 32P-labeled ATP during protein phosphorylation by protein kinases, or chemically by modification of amino acid side chains. (Kelman et al., 1995)

35S-Methionine/Cysteine labeling of Proteins

One of the most widely used radiolabels for proteins is 35S, due to its low-energy beta emissions which cause very little damage to the cells. 35S is sufficiently radioactive (∼40 TBq/mmol), therefore even modest incorporation can lead to measurable accumulation of labeled material while also being readily detectable.

The labeling /incubation time for incorporation of the 35S label is relatively small.  35S label is also quite energetic and it is not impeded by media such as polymerized acrylamide and can therefore be used successfully in autoradiography of 1D or 2D gel electrophoresis.

35S can be introduced into proteins as 35S-methionine/cysteine. Methionine is an essential amino acid containing sulfur, therefore using 35S-methionine ensures that the radiolabel rapidly and effectively enters the protein biosynthetic pathway.  Further, using methionine ascertains that at least one 35S-methionine is incorporated into every new protein via its start codon, ensuring all new proteins are radiolabeled.

To study protein synthesis, cells are typically incubated in a methionine-free media with 35S-methionine during mRNA translation (few hours to overnight). Optimum incubation time may vary depending on the individual proteins. Then, the newly synthesized protein can be identified from the incorporation of 35S-methionine by autoradiography after immunoprecipitation with a specific antibody and separation on a polyacrylamide denaturing gel.

32P-Orthophosphate labeling of Proteins

Radiolabel 32P is a beta emitter that is widely used to study the phosphorylation of proteins. In this method, radiolabeled phosphate is incorporated into the endogenous cellular ATP pool from which it is then utilized in various physiological pathways including phosphorylation.

Efficient 32P labeling of proteins can be attained by labeling the cells in a phosphate-free media for an extended period (a few hours to overnight). If the cells are incubated longer with the radiolabel it might result in the radiolabeling of the DNA and RNA. This problem can be overcome through purification by immunoprecipitation which will remove the DNA and RNA molecules from the proteins.

In Vitro Radiolabeling of Proteins

In vitro or cell-free assays can be performed for cytotoxic proteins or proteins with high enzymatic activity or other features which make protein synthesis difficult in cell culture. A major advantage of cell-free protein synthesis is the production of large quantities of protein with very few contaminating cellular proteins and other degradation products that come from cellular preparations.

In vitro radiolabeling is a simple and straightforward method. The main requirement of cell-free protein synthesis is obtaining a high-purity DNA or RNA template for the protein of interest. The template is first added to the assay mix, which contains buffers, ribosomes, amino acids (including 35S-Met), and DNAse/RNAse inhibitors.  These components are mixed and incubated for 30 min to 2 hours (depending on the commercial kit being used and the protein of interest). Finally, the newly-synthesized proteins are separated by polyacrylamide gel (under reducing conditions) and visualized using autoradiography or phosphorimaging.


Radioimmunoassays are highly specific and sensitive assays that employ antibodies to detect and quantitate the amount of antigen in a sample. The high sensitivity of these assays allows us to detect antigen concentrations as low as a few picograms when using antibodies of high affinity (Kd = 10-8 - 10-11 M).

Radioimmunoassay is based on the principle of competitive binding, where a radioactive antigen ("tracer") competes with a non-radioactive antigen for a fixed number of antibody or receptor binding sites. When unlabeled antigen from (standards or samples) and a fixed amount of tracer (radiolabeled protein antigen) are allowed to react with a constant and limiting amount of antibody, decreasing amounts of radiolabelled tracer are bound to the antibody as the amount of unlabeled antigen is increased.

125I and 3H are commonly used radiolabel for radioimmunoassays. Following incubation, the separation of the antibody-antigen complexes from free antigen is accomplished by precipitation of the antibody-bound radiolabeled tracer with either a) a secondary antibody solution directed against the genus or species-specific immunoglobulins of the primary antibody, or b) by use of polyethylene glycol.  Both these precipitation methods require the presence of a carrier immunoglobulin.

After centrifugation, the supernatant containing the unbound antigen is decanted (into an appropriate radioactive liquid waste tray), and the pellet containing the antibody-antigen complex is counted in a scintillation counter.  A standard dose-response curve is generated from the standards and the unknowns are calculated by interpolation.

With suitable precautions in place, there are numerous applications where radiolabelled proteins are used because of their specificity and sensitivity.


  • KELMAN, Z., NAKTINIS, V. & O'DONNELL, M. 1995. Radiolabeling of proteins for biochemical studies. Methods Enzymol, 262, 430-42.
  • STEINBERG, R. A. 1983. Radiolabeling and detection methods for studying metabolism of regulatory subunit of cAMP-dependent protein kinase I in intact cultured cells. Methods Enzymol, 99, 233-43.
  • SUGIURA, G., KUHN, H., SAUTER, M., HABERKORN, U. & MIER, W. 2014. Radiolabeling strategies for tumor-targeting proteinaceous drugs. Molecules, 19, 2135-65.

Further Reading

Last Updated: Apr 8, 2021


The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of AZoLifeSciences.
Post a new comment
Azthena logo powered by Azthena AI

Your AI Assistant finding answers from trusted AZoM content

Your AI Powered Scientific Assistant

Hi, I'm Azthena, you can trust me to find commercial scientific answers from

A few things you need to know before we start. Please read and accept to continue.

  • Use of “Azthena” is subject to the terms and conditions of use as set out by OpenAI.
  • Content provided on any AZoNetwork sites are subject to the site Terms & Conditions and Privacy Policy.
  • Large Language Models can make mistakes. Consider checking important information.

Great. Ask your question.

While we only use edited and approved content for Azthena answers, it may on occasions provide incorrect responses. Please confirm any data provided with the related suppliers or authors. We do not provide medical advice, if you search for medical information you must always consult a medical professional before acting on any information provided.

Your questions, but not your email details will be shared with OpenAI and retained for 30 days in accordance with their privacy principles.

Please do not ask questions that use sensitive or confidential information.

Read the full Terms & Conditions.