| Assay Method Information | |
| | Biochemical Kinase Assay |
| Description: | First, 250 nL of compound dissolved in DMSO (100-fold of the desired concentration) was dispensed into a 384-well plate. A 12.5 μL substrate solution containing ATP (2 mM) and fluorogenic phosphorylation substrate AQT0101 (26 M for ALK and ROS, AssayQuant) or AQT0104 (26 μM for TRKB, AssayQuant) in buffer (50 mM HEPES pH 7.5, 0.0100 Brij-35, 0.5 mM EGTA, 10 mM MgCl2) was added and mixed thoroughly. Then, a 12.5 μL kinase solution containing ALK (1.5 nM, Carna, 08-518), ALK I1171N/D1203N (4 nM, SignalChem, custom-made), ROS1 (0.6 nM, Carma, 08-163), ROS1-G2032R (0.5 nM, SignalChem, R14-121BG), or TRKB-wt (1.5 nM, SignalChem, N17-11G) kinase domains in buffer (50 nM HEPES pH 7.5, 0.01% Brij-35,200 glycerol, 0.4 mg/mL BSA, 0.5 mM EGTA, and 10 mM MgCl2) was added and mixed thoroughly. The plate was sealed and read by SpectraMax Paradigm at λ=485 nm every 2 minutes for 120 minutes at 30° C. Exemplary data is given in Table 3. Initial rates of reaction (v) were calculated from the change in fluorescence intensity over time during the initial, linear portion of the reaction. |
| Affinity data for this assay | |
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