| Assay Method Information | |
| | Binding Activity Against Human TrkA Protein |
| Description: | Measurements were made using TrkA LanthaScreen™ Eu Kinase Binding Assay (ThermoFisher SCIENTIFIC). Into 384 well plates (Corning), a 2.5 μL solution having each concentration of the test compound (1) diluted in Kinase buffer (ThermoFisher SCIENTIC) and a 2.5 μL having 15 nM TrkA enzyme (ThermoFisher SCIENTIFIC) were added. In addition, a 5 μL having 3 nM Eu-anti-His Tag antigen (ThermoFisher SCIENTIFIC) and 5 μL having 30 nM Kinase™ Tracer 236 (ThermoFisher SCIENTIFIC) were added, and allowed to react at room temperature for 60 minutes. After the reaction, the fluorescence ratio was calculated as the binding amount of the test compound (1) with TrkA enzyme, by measuring the fluorescence intensity (Emission wavelength 615 nm) and TR-FRET (Emission wavelength 665 nm) of Europium by the excitation wavelength of 340 nm using EnVision 2100 (PerkinElmer). The inhibitory activity (IC50 value) of the test compound (1) was calculated as 0% fluorescence ratio of the wells to which solvent was added instead of the test compound (1) and as 100% fluorescence ratio of the wells to which no TrkA protein was added. |
| Affinity data for this assay | |
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