Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Assay of protein kinases using radiolabeled ATP: a protocol

Nature Protocols volume 1, pages 968–971 (2006)Cite this article

Abstract

Protein kinase activity results in the incorporation of radiolabeled phosphate from [γ-32P]ATP into a peptide or protein substrate. The measurement of the amount of radioactivity incorporated into a substrate as a function of time and enzyme concentration allows enzyme activity to be quantified. The activity is expressed as a 'unit', where 1 unit corresponds to the amount of protein kinase that catalyzes the incorporation of 1 nanomole of phosphate into the standard substrate in 1 minute. Specific activity is defined as units of activity per milligram protein. The assay format described here is quick, simple, inexpensive, sensitive and accurate, provides a direct measurement of activity and remains the 'gold standard' for the quantification of protein kinase activity. Up to 40 samples can be assayed manually at one time, and the assay takes one person less than 1 hour to complete.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Cohen, P. Protein kinases, the major drug targets of the 21st century? Nat. Rev. Drug Discov. 1, 309–316 (2002).

    Article  CAS  Google Scholar 

  2. Witt, J.J. & Roskoski, R., Jr. Rapid protein kinase assay using phosphocellulose-paper absorption. Anal. Biochem. 66, 253–258 (1975).

    Article  CAS  Google Scholar 

  3. Glass, D.B., Masaracchia, R.A., Feramisco, J.R. & Kemp, B.E. Isolation of phosphorylated peptides and proteins on ion exchange papers. Anal. Biochem. 87, 566–575 (1978).

    Article  CAS  Google Scholar 

  4. Roskoski, R., Jr. Assays of protein kinase. Methods Enzymol. 99, 3–6 (1983).

    Article  CAS  Google Scholar 

  5. Wei, Y.F. & Matthews, H.R. A filter-based protein kinase assay selective for alkali-stable protein phosphorylation and suitable for acid-labile protein phosphorylation. Anal. Biochem. 190, 188–192 (1990).

    Article  CAS  Google Scholar 

  6. Tan, E., Lin Zu, X., Yeoh, G.C., Besant, P.G. & Attwood, P.V. Detection of histidine kinases via a filter-based assay and reverse-phase thin-layer chromatographic phosphoamino acid analysis. Anal. Biochem. 323, 122–126 (2003).

    Article  CAS  Google Scholar 

  7. Park, Y.W. et al. Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence. Anal. Biochem. 269, 94–104 (1999).

    Article  CAS  Google Scholar 

  8. Wu, J.J. Comparison of SPA, FRET and FP for kinase assays. Methods Mol. Biol. 190, 65–85 (2002).

    CAS  PubMed  Google Scholar 

  9. Seethala, R. & Menzel, R. A homogeneous, fluorescence polarization assay for src-family tyrosine kinases. Anal. Biochem. 253, 210–218 (1997).

    Article  CAS  Google Scholar 

  10. Turek-Etienne, T.C. et al. Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease, and 1 phosphatase. J. Biomol. Screen. 8, 176–184 (2003).

    Article  CAS  Google Scholar 

  11. Davies, S.P., Reddy, H., Caivano, M. & Cohen, P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95–105 (2000).

    Article  CAS  Google Scholar 

  12. Bain, J., McLauchlan, H., Elliott, M. & Cohen, P. The specificities of protein kinase inhibitors: an update. Biochem. J. 371, 199–204 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the UK Medical Research Council, The Royal Society, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck and Co., Merck KGaA and Pfizer for financial support.

Author information

Author notes

  1. C James Hastie and Hilary J McLauchlan: These authors contributed equally to this manuscript.

Authors and Affiliations

  1. Division of Signal Transduction Therapy, Medical Sciences Institute–Wellcome Trust Biocentre Complex, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland

    C James Hastie & Hilary J McLauchlan

  2. Medical Research Council Protein Phosphorylation Unit, Medical Sciences Institute–Wellcome Trust Biocentre Complex, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland

    Philip Cohen

Authors
  1. C James Hastie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hilary J McLauchlan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Philip Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Philip Cohen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hastie, C., McLauchlan, H. & Cohen, P. Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc 1, 968–971 (2006). https://doi.org/10.1038/nprot.2006.149

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.149

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing