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 affinity and efficacy of potential inhibitors. Here, we describe protocols for heterologous expression and purification of JAK kinases from insect cells, assays to determine the activity of these purified kinases, and finally inhibition assays to determine the effectiveness of potential inhibitors.
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Acknowledgements
This work was made possible through Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC IRIISS. This research was supported by an NHMRC Program Grant (461219), NIH Grant (CA022556), NHMRC CDA (JJB, 516777), ARC Future Fellowship (JMM, FT100100100), and NHMRC Project Grant (1011804).
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Babon, J.J., Murphy, J.M. (2013). In Vitro JAK Kinase Activity and Inhibition Assays. In: Nicholson, S., Nicola, N. (eds) JAK-STAT Signalling. Methods in Molecular Biology, vol 967. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-242-1_3
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DOI: https://doi.org/10.1007/978-1-62703-242-1_3
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