| Assay Method Information | |
| | FGFR Biochemical Assay |
| Description: | The following procedure was used to test the compounds described herein:Dilute the test compounds to 100× of the final desired highest concentration in reaction by 100% DMSO.Perform a serial dilution in a 384-well source plate by transferring 15 μL to 30 μL of 100% DMSO in the next well and so forth for a total of 10 concentrations.Dispense 100 nL of compound solution each well from the source plate to a 384-well assay plate in duplicates by Echo (Labcyte).Prepare a solution of 2×FGFR enzyme (Carna) in kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 4 mM DTT, 0.01% Tween-20 and 0.01% BSA). The final concentration of FGFR1/2/3/4 is 0.01. 0.01, 0.1 and 1 nM, respectively.Add 5 μL of kinase solution to each well of the compound-containing assay plate, except for low control wells without enzyme (add 5 μL of 1× kinase buffer instead).Shake the plate and incubate at room temperature for 10 minutes (or 60 min as indicated as * in table 1).Prepare the substrate solution of Fluorescein-Poly GT (Invitrogen) and ATP (Sigma) in 1× kinase reaction buffer at 2-fold of the final concentration of each reagent desired in the assay (final concentration of ATP is 28, 22, 35 and 238 μM for FGFR1/2/3/4 respectively; Fluorescein-Poly GT is fixed at 20 nM).Add 5 μL of substrate solution to each well of the assay plate to initiate the kinase reaction.Shake the plate shortly and incubate at room temperature for 30 minutes.Prepare 2× detection solution in Antibody Dilution Buffer (LanthaScreen® Tb-PY20 Antibody Kit, Invitrogen).Add 10 μL of detection solution to each well of the assay plate to stop the reaction.Mix briefly with centrifuge and incubate at room temperature for 60 minutes before reading on a microplate reader.Collect data on Envision with excitation at 340 nm and emission at 520 nm and 495 nm. |
| Affinity data for this assay | |
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