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Assay of protein kinases using radiolabeled ATP: a protocol

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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.

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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.

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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
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  2. Hilary J McLauchlan
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  3. Philip Cohen
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Correspondence to Philip Cohen.

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The authors declare no competing financial interests.

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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

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