| Assay Method Information | |
| | Kinase Inhibitory Activity |
| Description: | Inhibitory activities of the compound of the present application on three kinases TRKa, TRKA(G595R) and TRKC(G623R) were measured.2. Experimental Materials2.2.1 Reagents and ConsumablesName of reagent Supplier Article number Batch numberTRKa Carna 08-186 13CBS-0565GTRKA (G595R) signalchem N16-12BG-100 H2714-7TRKC(G623R) signalchem N18-12CH-100 D2567-8Kinase substrate 22 GL 112393 P190329-SL112393DMSO Sigma D8418-1L SHBG3288V384-well plate PerkinElmer 6007290 810712(white)2.2.2 InstrumentsCentrifuge (manufacturer: Eppendorf, model number: 5430); microplate reader (manufacturer: Perkin Elmer, model number: Caliper EZ ReaderII); and Echo 550 (manufacturer: Labcyte, model number: Echo 550)3. Experimental Method3.1 A tested compound was accurately weighed and dissolved in 100% DMSO to prepare into a 10 mM solution.3.2 Kinase Reaction Process3.2.1 1×kinase buffer was prepared.3.2.2 Preparation of compounds of gradient concentrations: the tested compound had an initial concentration of 1000 nM, and was diluted into a 100% DMSO solution of 100 times final concentration in the 384-well plate, and then the 100% DMSO solution was diluted by 3 times to obtain DMSO solutions of the compound with 10 concentrations. 250 nL of the compound of 100 times final concentration was transferred to a reaction plate OptiPlate-384F by a dispenser Echo 550.3.2.3 A kinase solution of 2.5 times final concentration was prepared with 1×kinase buffer.3.2.4 10 μL of kinase solution of 2.5 times final concentration was added into a compound well and a positive control well respectively; and 10 μL of 1×kinase buffer was added into a negative control well.3.2.5 The reaction plate was shaken and mixed evenly, and then incubated at room temperature for 10 minutes.3.2.6 Mixed solutions of ATP and kinase substrate 22 of 5 times and 3 times final concentrations were prepared with 1×kinase buffer.3.2.7 15 μL of mixed solution of ATP and substrate of 5 times or 3 times final concentration was added.3.2.8 The 384-well plate was centrifuged at 1,000 rpm for 30 seconds, shaken and mixed evenly, and then incubated at room temperature for corresponding time.3.2.9 The kinase reaction was terminated.3.2.10 A conversion rate was read by the microplate reader Caliper EZ Reader. |
| Affinity data for this assay | |
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