Analysis of Janus Tyrosine Kinase Phosphorylation and Activation

  • Jeremy A. Ross
  • Georgialina Rodriguez
  • Robert A. KirkenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 967)


Activation of Janus kinases (Jaks) occurs through autophosphorylation of key tyrosine residues located primarily within their catalytic domain. Phosphorylation of these tyrosine residues facilitates access of substrates to the active site and serves as an intrinsic indicator of Jak activation. Here, we describe the methods and strategies used for analyzing Jak phosphorylation and activation. Tyrosine-phosphorylated (active) Jaks are primarily detected from cell extracts using anti-phosphotyrosine-directed Western blot analysis of Jak-specific immunoprecipitates. Additionally, receptor pull-down and in vitro kinase assays can also be utilized to measure cellular Jak catalytic activity. In addition to tyrosine phosphorylation, recent evidence indicates Jaks can be serine phosphorylated upon cytokine stimulation, however the lack of commercially available antibodies to detect these sites has hindered their analysis by Western blot. However, phosphoamino acid analysis (PAA) has been employed to monitor Jak serine and threonine phosphorylation. Over the past decade, remarkable advances have been made in our understanding of Jak function and dysfunction, however much remains to be learned about their complex regulatory mechanisms.

Key words

Janus kinase (Jak) Tyrosine phosphorylation Immunoprecipitation SDS-PAGE Immunoblot Kinase assay Phosphoamino acid analysis (PAA) 



This work was supported by grants from the Lizanell and Colbert Coldwell Foundation, Edward N. and Margaret G. Marsh Foundation, and grant G12MD007592 from the National Center on Minority Health and Health Disparities (NCMHD), a component of the National Institutes of Health (NIH).


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jeremy A. Ross
    • 1
    • 2
  • Georgialina Rodriguez
    • 1
    • 2
  • Robert A. Kirken
    • 1
    • 2
    Email author
  1. 1.Department of Biological SciencesThe University of Texas at El PasoEl PasoUSA
  2. 2.Border Biomedical Research CenterThe University of Texas at El PasoEl PasoUSA

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