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| | FlashPlate Assay |
| Description: | The kinase assay was carried out as 384-well FlashPlate assay. As test plates 384-well streptavidine coated FlashPlate microtitre plates from Perkin Elmer (USA) were used. The components of the kinase reaction were pipetted into the assay plate. 4.5 nM of GST tagged human recombinant RON kinase (Life technologies), 500 nM of biotinylated peptide substrate RDILDREYYSVQQHRH-amide (autophosphorylation site derived peptide substrate, custom-made) and 2 μM of ATP (with 0.5 μCi of <33>P-ATP/well) were incubated in a total volume of 50 μl (50 mM of HEPES, 5 mM of MgCl2, 2 mM of DTT, 0.1% of BSA, 0.01% Igepal CA630, 1% DMSO, pH 7.5) in the presence or absence of test substance (10 concentrations) at 22 [deg.] C for 30 min. The reaction was stopped using 50 μl of 200 mM EDTA solution. After incubation for a further 80 min at room temperature, the supernatants were removed by suction, and the wells were washed three times with 100 μl of 0.9% NaCl solution each time. |
| Affinity data for this assay | |
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