| Assay Method Information | |
| | ADP-Glo Kinase assay |
| Description: | An inhibitory effect of the inventive compound on JAK was identified as follows.A control material and a test material were prepared through dilution at each concentration by using DMSO. At the same time, ATP (250 uM) and JAK's substrate (JAK1, IRS-1tide 40 ng/mL) were prepared through dilution in kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, the substrate, the ATP and JAK enzymes were mixed in an eppendorf tube, and then subjected to reaction in an incubator at 30° C. for 40 minutes.ADP-GloFigure US12180185-20241231-P00001 reagent included in ADP-Glo™ Kinase Enzyme System (Promega, USA, V9571) was added to each eppendorf tube, and then subjected to reaction in the incubator at 30° C. for 40 minutes.A kinase detection reagent included in the ADP-Glo™ Kinase Enzyme System was inserted into the eppendorf tube, after which luminescence was measured by using Wallac Victor 2TM with an integration time set to 1 second, such that an inhibitory capacity of the test material on JAKs phosphorylation was analyzed. A concentration of the compound, at which JAK enzyme activity inhibition occurs 50% compared to the control group, was determined as IC50 (nM) of an inhibitor. |
| Affinity data for this assay | |
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