| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence (HTRF) |
| Description: | Compound preparation: Add 45 μL of DMSO to 5 μL of stock solution at a concentration of 10 mM to prepare the solution LY2874455 at 1000 M; then add 12 μL of the compound at 1000 μM to 88 L of DMSO to prepare the solution LY2874455 at 120 μM as the starting concentration; add 48 μL of DMSO to 2 μL of stock solution at a concentration of 10 mM to prepare the solution BLU554 at 400 μM as the starting concentration; add 15 μL of DMSO to 10 μL of sample compound stock solution at a concentration of 10 mM to prepare a sample compound solution at 4 mM as the starting concentration; transfer the compound solution to the target plate with the dispenser Echo.Kinase reaction: Add 5 μL of kinase solution to each well, incubate the compound and kinase at room temperature for 60 min, add 5 μL of substrate and ATP mixture to start reacting at 37° C. for a certain period of time (30 min for FGFR1 kinase test, and 40 min for FGFR4 kinase test), and then add 10 μL of X1665 and antibody detection reagent mixture to stop; after 60 min of incubation at room temperature, read the TR-FRET signal with microplate reader SPARK 10M at the transmit wavelength of 665 nM/612 nM. Test enzyme activity at 10 concentrations for each compound, and calculate the IC50 value of the compound with the data with the analysis software. |
| Affinity data for this assay | |
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