title: In vitro JAK kinase activity and inhibition assays.
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.
authors.affiliation: Cancer and Haematology/Structural Biology Divisions, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia. babon@wehi.edu.au
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Babon
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D057075:Enzyme Assays / Q000379:methods*; D015870:Gene Expression; D053612:Janus Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011874:Radiometry; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D047428:Protein Kinase Inhibitors; D053612:Janus Kinases