| Assay Method Information | |
| | Enzyme Assay |
| Description: | he potency of compounds inhibiting human isoforms of FGFR kinase was determined using Life Technologies' Homogeneous Time Resolved Fluorescence (HTRF)-based binding assay technology. An incubation was conducted with either 5 nM dephosphorylated FGFR1 (Array Biopharma, p1702; SEQ ID NO: 1, amino acids 458 to 765, dephosphorylated by co-expression with PTP1b (protein tyrosine phosphatase 1B)), 5 nM dephosphorylated FGFR2 (Life Technologies, Cat. No. PV4106 that had been dephosphorylated with Lambda protein phosphatase (New England Biolabs, cat #P0753)) or 5 nM phosphorylated FGFR3 (Array Biopharma, p1836; SEQ ID NO: 5, amino acids 449 to 759), 50 nM Kinase Tracer 236 (Life Technologies Cat. No. PR9078A), 2 nM Biotin-anti-6HIS (Life Technologies Cat. No. PV6090) and 2 nM Europium-Streptavidin (Life Technologies Cat. No. PV6025) along with test compound in a buffer consisting of 50 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5), 5 mM MgCl2, 0.005% Triton X-100, 1 mM DTT, 1 mM NaVO4 and 2% DMSO in a final volume of 12 μL. Compounds were typically prepared as a 3-fold or 4-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60 minute incubation at 22° C., the extent of tracer displacement was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. One hundred POC was determined using no test compound, and 0 POC was determined in the presence of 1 μM of an appropriate control inhibitor. A 4-parameter logistic curve was fit to the POC values as a function of the concentration of compound, and the IC50 value was the point where the best-fit curve crossed 50 POC. |
| Affinity data for this assay | |
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