| Assay Method Information | |
| | JAK2 Activity Inhibition Assay |
| Description: | The enzymatic activity of JAK2 was assessed by detecting the substrate phosphorylation level in a kinase reaction with a homogeneous time-resolved fluorescence (HTRF) kinase detection kit (Cisbio, 62TK0PEC). A reaction buffer contained an enzyme buffer (1×), 5 mM MgCl2, 1 mM DTT and 0.01% Brij35 from the kit; a human recombinant JAK2 protein (Carna Biosciences, 08-045) was diluted to a kinase reaction solution of 0.15 ng/μL with the reaction buffer; a substrate reaction solution contained 2.5 μM ATP and a biotin-labeled tyrosine kinase substrate diluted to 0.25 μM with the reaction buffer; a detection buffer contained 0.1 ng/μL Eu3+ labeled cage antibody (Cisbio, 61T66KLB) and 12.5 nM streptavidin-labeled XL665 (Cisbio, 610SAXLB) in the reaction buffer; the compound was dissolved to 10 μM in DMSO, followed by a serial 4-fold dilution with DMSO to a minimum concentration of 0.061 nM. Each concentration was further diluted 40-fold with the reaction buffer. To a 384-well assay plate (Corning, 3674) were added 4 μL of the compound solutions having a series of concentrations and 2 μL of JAK2 kinase reaction solutions. After being mixed evenly, the mixtures were incubated at room temperature for 15 minutes, and then 4 μL of the substrate reaction solutions were added. The reaction mixtures were incubated at room temperature for 30 minutes. Then the reaction mixtures were added with 10 μL of detection solutions, mixed evenly, and allowed to stand at room temperature for 30 minutes. An Envision plate reader (Perkin Elmer) was then used to measure the progress of the reaction at wavelengths of 620 nm and 665 nm. |
| Affinity data for this assay | |
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