Assay of protein kinases using radiolabeled ATP: A protocol

C. James Hastie, Hilary J. McLauchlan, Philip Cohen (Lead / Corresponding author)

    Research output: Contribution to journalArticlepeer-review

    168 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)968-971
    Number of pages4
    JournalNature Protocols
    Volume1
    Issue number2
    DOIs
    Publication statusPublished - 1 Jul 2006

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