Authors
Azam Mohammad and George Daley
Summary
Protein kinases are one of the largest families of the protein coding genes: constituting some 2% of the expressed proteins, regulate almost all biochemical pathways and phosphorylate up to 30% of the proteome. More than 400 human diseases have been linked, directly or indirectly, to protein kinases. Deregulated tyrosine kinases have been implicated in neoplastic development. Here, we describe biochemical procedures to monitor the activation of the tyrosine kinases (ABL, SRC, PDGFRA, PDGFRB and EGFR). These kinases and its constitutively activated variants were expressed in mammalian cells and kinase activity was measured by tyrosine phosphorylation by immunoblot analysis. We also describe the procedure for cellular transformation of BAF3 cells for IL-3 independent cell proliferation, and the development of leukemia in mice. The combination of these procedures is useful to evaluate the activating mutations in kinases and its potential to develop cancer. This protocol can be completed in 3-4 months.Further details
The protocol was published on Protocol Exchange in 2008. To see the entire protocol, click on the source link.Advertisement
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