| Assay Method Information | |
| | Kinase Enzyme Assays |
| Description: | The compounds of the invention were screened in vitro for their ability to inhibit c-Met kinase activity. Briefly, histidine-tagged c-Met catalytic domain fusion protein (Invitrogen, # PV3143) was used for the assay. IC50 measurements were based on the degree of phosphorylation of poly Glu-Tyr (Sigma-Aldrich, # P0275) that was coated (0.01 mg/per well) on 96-well microplates (R&D systems, # DY990). The reaction was carried out in a 50 μL solution containing 50 mM HEPES (pH 7.5), 10 mM MnCl2, 10 mM MgCl2, 0.5 mM DTT, 100 μM Na3VO4, 5 μM ATP (Cell Signaling Technology, #9804) and serial dilutions of the test compound. The reaction lasted for 25 minutes at 30° C. After the reaction was completed, the contents of the plates were discarded. Plates were then washed with TBS-T (250 μL/well, 5×) and then blocked with TBS-T containing 1% BSA for 2 hours. The contents of the plates was discarded, and 100 μL (per well) of peroxidase-labeled anti-phospho-tyrosine antibody (Sigma, # A5964) diluted (1:60,000) in 1% BSA containing TBS-T were then added and incubated for 1 hour, Plates were washed with TBS-T (250 μL/well, 5×) and followed by the color reaction using 100 ML (1:1 mixture) of H2O2 and tetramethylbenzidine (R&D Systems, # DY999). The reaction was stopped in minutes with 100 ML of 2 N H2SO4. The optical density was measured immediately using a microplate reader at 450 nm with wavelength correction at 540 nm. |
| Affinity data for this assay | |
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