PIM3 Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
Pim kinases are constitutively active serine/threonine kinases that promote growth factor-independent proliferation by phosphorylation and subsequent inhibition of a range of cellular proteins. Activity of Pim kinases is physiologically regulated via protein stability. Especially Pim1 and Pim2 kinase have been found to be overexpressed or mutated in a variety of human tumors including B cell lymphomas, leukemias, prostate cancer and oral cancer.
Transiently transfected Human embryonic kidney cells (HEK) express full length Pim3 kinase as well as Pim-substrate Bad labeled with a myc-tag. Constitutive activity of exogenous Pim3 results in phosphorylation of exogenous Bad at Ser112. Upon compound incubation, cells are lysed and Bad phosphorylation is quantified by Sandwich-ELISA technique. The assay is validated based on the cognate inhibitor of Pim kinases AZD1208 (see Fig.1).
Substrate phosphorylation as a readout of intracellular kinase activity via ELISA
More information can be found on our website Cellular Phosphorylation Assay Services.
Reference compound IC50 for PIM3
Cognate Pim inhibitor AZD1208 blocks Pim3 and inhibits phosphorylation of Bad at Ser112 with highly reproducible IC50 values. The graph shows a representative result.