MET Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
The proto-oncogene MET is a receptor tyrosine kinase also known as hepatocyte growth factor receptor. The primary single chain precursor protein is post-translationally cleaved to produce the alpha and beta subunits, which are disulfide linked to form the mature receptor. The beta-subunit of Met is the cell-surface receptor for hepatocyte growth factor (HGF). MET is a membrane receptor that is essential for embryonic development and wound healing. MET and its ligand HGF are overexpressed in several human cancers and are amplified during the transition between primary tumors and metastasis.
c-MET, HGFR, RCCP2
The human gastric adenocarcinoma cell line MKN45 is known to overexpress MET. MET overexpression results in a constitutive, ligand-independent autophosphorylation of the kinase. By adding SU11274 phospho-MET levels are largely decreased and thus the dynamic behaviour to determine inhibitory potentials of compounds was achieved. Phospho-MET signal is subsequently quantified by Sandwich-ELISA technique. The assay is validated based on known inhibitors of MET kinase activity (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 MET
PHA665752 and SU11274 are known to inhibit the phospho-MET signal in a highly specific manner. Both compounds were included in the validation process and the cellular MET assay generated reproducible IC50 values. The graphs show representative results.