| Assay Method Information | |
| | JAK2 Kinase Domain Enzymatic Activity Assay |
| Description: | A reaction buffer containing the following components: an enzyme buffer (1×), 5 mM MgCl, 1 mM DTT and 0.01% Brij35 from the kit; a human recombinant JAK2 kinase domain protein (Carna Biosciences, Cat. No. 08-045) diluted to a solution of 0.15 ng/μL with the reaction buffer; a substrate reaction solution containing 2.5 μM ATP and a biotinylated tyrosine kinase substrate diluted to 0.25 μM with the reaction buffer; a detection solution containing 0.1 ng/μL Eu labeled cage antibody (Cisbio, Cat. No. 61T66KLB) and 12.5 nM streptavidin-labeled XL665 (Cisbio, Cat. No. 610SAXLB) in the reaction buffer. The test compound is dissolved to 1 mM in DMSO, followed by a serial 4-fold dilution with DMSO to a minimum concentration of 61 nM. Each concentration is further diluted 40-fold with the reaction buffer. To a 384-well assay plate (Corning, Cat. No. 3674) are added 4 μL of compound solution and 2 μL of JAK2 kinase solution. The mixture is incubated at room temperature for 15 minutes, and then added with 4 μL of the substrate reaction solution. After further incubation at room temperature for 30 minutes, the reaction mixture is added with an equal volume of 10 μL detection solution and allowed to stand at room temperature for 30 minutes. An Envision plate reader (Perkin Elmer) is then used to measure the progress of the reaction at 620 nm and 665 nm. The ratio of absorbances at 665 nm and 620 nm is positively correlated with the degree of substrate phosphorylation, therefore the activity of JAK2 kinase is detected. |
| Affinity data for this assay | |
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