Assaying protein kinase activity with radiolabeled ATP

Aroon S. Karra, Steve Stippec, Melanie H. Cobb

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [γ-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Original languageEnglish (US)
Article numbere55504
JournalJournal of Visualized Experiments
Volume2017
Issue number123
DOIs
StatePublished - May 26 2017

Fingerprint

Adenosinetriphosphate
Protein Kinases
Assays
Phosphotransferases
Adenosine Triphosphate
Proteins
Cell signaling
Sequence Deletion
Cell culture
Visualization
Cell Culture Techniques
Pharmacology
Defects
Testing
Substrates
Neoplasms
Therapeutics

Keywords

  • Biochemical assay
  • Biochemistry
  • Issue 123
  • Protein kinase
  • Radiolabeled assay
  • Signal transduction

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Assaying protein kinase activity with radiolabeled ATP. / Karra, Aroon S.; Stippec, Steve; Cobb, Melanie H.

In: Journal of Visualized Experiments, Vol. 2017, No. 123, e55504, 26.05.2017.

Research output: Contribution to journalArticle

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