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| | HTRF kinase assay |
| Description: | The c-Met kinase activity was evaluated by homogeneous time-resolved fluorescence (HTRF) assays. In a 384-well plate, 20 µg/mL (Glue, Tyr) 4:1 was added, followed by the addition of 50 µL of 10 mM ATP solution diluted in kinase reaction buffer (50 mM HEPES, pH 7.0, 1 M DTT, 1 M MgCl2, 1 M MnCl2, and 0.1% NaN3) into each well. Various concentrations of compounds diluted in 10 µL of 1% DMSO (v/v) are used as the negative control. The reaction was initiated by the addition of purified c-Met kinase diluated in 39 µL of kinase reaction buffer solution. The reaction was incubated for 30 min at 25 °C and stopped by the addition of 5 µL of Streptavidin-XL665 and 5 µL Tk Antibody Cryptate working solution to all of the wells. The plate was read at 320 nm and 615 nm. |
| Affinity data for this assay | |
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