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 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 ..