| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence (HTRF) Assay |
| Description: | JAK2: A testing platform for JAK2 kinase activity was established based on Homogeneous Time-Resolved Fluorescence (HTRF) assay, and the activities of the compounds were tested using the platform. The compounds were subjected to three-fold gradient dilution with 100% DMSO with a starting concentration of 1 mM (11 concentrations in total). 4 μL of diluted sample for each concentration was added to 96 μL of reaction buffer (50 mM HEPES, pH7.4, 10 mM MgCl2, 1 mM EDTA, 0.01% Tween-20, 0.005% BSA and 2 mM DTT) and mixed homogeneously. 2.5 μL of the resulting liquid was then added to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), and 5 μL of JAK2 kinase (purchased from Carna, and the final concentration thereof is 0.05 nM) was added, and mixed homogeneously by centrifugation. Then 2.5 μL of a mixture of ATP (the final concentration is 5 μM) and TK peptide (HTRF KinEASE -TK, purchased from Cisbio, and the final concentration is 100 nM) was added to initiate the reaction (the total reaction volume is 10 μL). The 384-well plate was placed in an incubator to incubate for 120 min at 23° C. Then the reaction was terminated by adding 5 μL of Eu3+ cryptate-labled anti-phosphotyrosine antibody (purchased from Cisbio), and 5 μL of Streptavidin-XL-665 (HTRF KinEASE -TK, purchased from Cisbio). After incubated for 1 hr in the incubator, the fluorescence values were read on Envision (purchased from PerkinElmer). |
| Affinity data for this assay | |
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