| Assay Method Information | |
| | JAK3 Kinase Inhibitory Activity Assay |
| Description: | (1) 1×Kinase buffer was prepared.(2) formulating compound concentration gradient: the test compounds (including the example compound and PF-06651600) were tested at a concentration of 10000 nM, 10-fold dilution, 10 concentrations, single-well detection. After being gradient dilution to a 100-fold final concentration, 250 nL of solutions were transferred to a 384-well plate using Echo550. 250 nL of 100% DMSO were added to each of the negative control wells and the positive control wells, respectively.(3) 2.5 times a final concentration of kinase solution was prepared with 1×Kinase buffer.(4) 10 μL of kinase solutions at 2.5 times were respectively added to the final concentration to the compound wells and positive control wells; and 10 μL of 1×Kinase buffer was added to the negative control wells.(5) The reaction mixtures were centrifuged at 1000 rpm for 30 seconds, shaken to mix, and incubated at room temperature for 10 minutes.(6) A mixed solution of 25/15 the final concentration of ATP and Kinase substrate 22 was prepared with 1×Kinase buffer.(7) The reaction was initiated by adding 15 μL of a mixed solution of 25/15 times the final concentration of ATP and substrate to the compound wells, positive control wells, and negative control wells of the 384-well plate.(8) The reaction mixtures in the 384-well plate were centrifuged at 1000 rpm for 30 seconds, shaken to mix, and incubated at room temperature for 30 minutes.(9) 30 μL of stop detection solution was added to stop the kinase reaction, and the reaction mixtures were centrifuged at 1000 rpm for 30 seconds, and shaken to mix.(10) The conversion rate was read by Caliper EZ Reader. 3. Data Analysis |
| Affinity data for this assay | |
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