| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | The inhibitory activity (IC50) of the amide compound for JAK1 and JAK2 under 1 mM ATP was measured by a mobility shift assay method. JAK1 was purchased from Carna Corporation (Cat. No. 08-144, Lot No. 11CBS-0144V) and JAK2 was purchased from Carna Corporation (Cat. No. 08-045, Lot No. 10CBS-0289R). JAK1 Peptide was purchased from GL (Cat. No. 758318, Lot No. P191104-TL758318) and Kinase substrate22 was purchased from GL (Cat. No. 112393, Lot No. P200403-CL112393). The positive control compound used was baricitinib. Specific steps are described below.1. A 1× Kinase buffer was formulated.2. Concentration gradients of the compound were formulated: a test compound with an initial concentration of 10000 nM (JAK1) or 30000 nM (JAK2) was diluted in a 384-well plate to a 100% DMSO solution with a 100-fold final concentration, wherein the compound was diluted by a fold of 3 to 10 concentrations. 250 nL of the compound with the 100-fold final concentration was transferred with a dispenser Echo 550 to the target plate.3. A kinase solution with a 2.5-fold final concentration was prepared with the 1× Kinase buffer.4. 10 μL of the kinase solution with the 2.5-fold final concentration was added to compound wells and positive control wells, separately; and 10 μL of 1× Kinase buffer was added to negative control wells.5. The plate was centrifuged at 1000 rpm for 30 s, shaken and uniformly mixed, and incubated for 10 min at room temperature.6. A mixed solution of ATP (the final concentration of ATP=1 mM) and Kinase substrate with a 5/3-fold final concentration was formulated with the 1× Kinase buffer.7. 15 μL of the mixed solution of ATP and the substrate with the 5/3-fold final concentration was added to initiate the reaction.8. The 384-well plate was centrifuged at 1000 rpm for 30 s, shaken and uniformly mixed, and incubated for respective times at room temperature.9. The kinase reaction was stopped by adding 30 μL of a detection termination solution, and the plate was centrifuged at 1000 rpm for 30 s and shaken and uniformly mixed.10. The conversion rate was read with Caliper EZ Reader and the half maximal inhibitory concentration (IC50) was calculated. |
| Affinity data for this assay | |
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