| Assay Method Information | |
| | TR-FRET (time-resolved fluorescence resonance energy transfer) Assay |
| Description: | TR-FRET (time-resolved fluorescence resonance energy transfer) method was used to detect the inhibitory activity of the compounds on FGFR1, FGFR2, FGFR3, FGFR4, and FGFR2 V564F kinase region fragments. The highest detection concentration of the compounds in 5 kinase experiments was 1000 nM, which was diluted in 3 folds, with a total of 11 concentrations (1000 nM to 0.017 nM). A kinase buffer (50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, and 0.01% Tween-20) was used to prepare 4× enzyme solution, 4× compound solutions diluted in gradient, and a 2×ATP/Peptide substrate solution. To a 384-well plate, 2.5 μL of enzyme solution and 2.5 μL of diluted 5× compound solution were added, and 5 μL of 2×ATP/Peptide substrate solution was then added. After reacting at room temperature for 60 minutes for FGFR1, FGFR2, FGFR3, and FGFR2 V564F kinases, and 120 minutes for FGFR4 kinase, 10 μL of a 2× termination and detection mixed liquid prepared with 1×LANCE detection buffer with an EDTA concentration of 20 mM and a Europium-anti-phosphotyrosine (PT66) concentration of 2 nM was added to each well of the plate. After reacting at room temperature for 60 minutes for FGFR1, FGFR2, FGFR3, and FGFR4, and 30 minutes for FGFR2 V564F, the fluorescence signal value in each well of the plate was measured by a microplate reader. |
| Affinity data for this assay | |
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