| Assay Method Information | |
| | Enzymatic Activity Assay |
| Description: | A testing platform for kinase activity of JAK2 (wild type and V617F mutant type) 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 dilutions with 100% DMSO with a starting concentration of 1 mM (11 dilutions in total). 4 μL of each dilution was added to 96 μL of reaction buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 0.005% BAS, 2 mM DTT) and mixed homogeneously. 2.5 μL of the resulting liquid was then added to a 384-well plate (OptiPlate-384, available from PerkinElmer), and then 5 μL of JAK2 kinase (available from Carna) was added. The mixture was mixed homogeneously by centrifugation. Then 2.5 μL of a mixture of ATP (the final concentration is the corresponding Km value) and TK peptide (HTRF KinEASE-TK, available from Cisbio) was added to initiate the reaction (the total reaction volumn is 10 μL). The 384-well plate was placed in an incubator and the reaction was allowed to conduct for 120 min at 23° C. Then the reaction was terminated by adding 5 μL of Eu3+ cryptate-labled anti-phosphotyrosine antibody (available from Cisbio), and 5 μL of Streptavidin-XL-665 (HTRF KinEASE-TK, available from Cisbio). The plate was incubated in the incubator for 1 hr, and then the fluorescence values were read on Envision (available from PerkinElmer). The excitation wavelength was 320 nm, and the emission wavelengths for detecton were 665 nm and 620 nm. The enzymatic activity was representd by a ratio of the two readout at the two emission wavelengths. |
| Affinity data for this assay | |
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