| Assay Method Information | |
| | Kinase Inhibitory Activity Assay |
| Description: | Kinase Reaction Process(1) 1×Kinase buffer was prepared.(2) Preparation of concentration gradient of compound: The tested compound has a test concentration starting from 1 μM, diluted 10 times, with 10 concentrations, and subjected to single-well or multi-well detection. The compound was diluted to a 100% DMSO solution with a final concentration of 100 times in a 384 source plate. 250 μL of the compound at a final concentration of 100 times was transferred to the target plate 384-well-plate using the pipette Echo 550.(3) A kinase solution with a final concentration of 2.5 times was prepared using 1× Kinase buffer.(4) 10 μL of the kinase solution with the final concentration of 2.5 times was added to a compound well and a positive control well, respectively; 10 μL of 1×Kinase buffer was added to a negative control well.(5) Centrifugation was conducted at 1000 rpm for 30 seconds, and the reaction plate was shaken and mixed well, and incubated at a room temperature for 10 minutes.(6) A mixed solution of ATP with a final concentration of 5/3 times and Kinase substrate 22 was prepared using 1×Kinase buffer.(7) A mixed solution of 15 μL of ATP with a final concentration of 5/3 times and a substrate was added for initial reaction.(8) The 384-well-plate was centrifuged at 1000 rpm for 30 seconds, shaken and mixed well, and incubated at a room temperature for 60 minutes.(9) 30 μL of a termination detection solution was added to stop the kinase reaction. Centrifugation was conducted at 1000 rpm for 30 seconds. Shaking and mixing well were conducted.(10) The conversion rate was read using Caliper EZ Reader. |
| Affinity data for this assay | |
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