In vitro JAK kinase activity and inhibition assays

Jeffrey J. Babon, James M. Murphy

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Otherpeer-review

16 Citations (Scopus)

Abstract

The discovery that a range of myeloproliferative diseases and leukemias are associated with Janus Kinase (JAK) mutations has highlighted the importance of JAK/STAT signalling in disease and sparked a renewed interest in developing JAK inhibitors. In vitro kinase assays are the most direct and quantitative method to assess mutant forms of JAK for altered enzymatic properties as well as verifying and quantifying the af finity and ef ficacy of potential inhibitors. Here, we describe protocols for heterologous expression and puri fication of JAK kinases from insect cells, assays to determine the activity of these puri fied kinases, and finally inhibition assays to determine the effectiveness of potential inhibitors.

Original languageEnglish
Title of host publicationJAK-STAT Signalling
Subtitle of host publicationMethods and Protocols
EditorsSandra E. Nicholson, Nicos A. Nicola
Place of PublicationTotowa NJ USA
PublisherHumana Press
Chapter3
Pages39-55
Number of pages17
Edition1st
ISBN (Electronic)9781627032421
ISBN (Print)9781627032414
DOIs
Publication statusPublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume967
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Activation
  • Enzyme
  • Enzyme kinetics
  • Inhibition assay
  • JAK
  • JAK1
  • JAK2
  • JAK3
  • Kinase
  • TYK2

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