| Assay Method Information | |
| | In Vitro Test of Activities of Jak1, Jak2, Jak3, Tyk2 Kinases |
| Description: | JAK3:Dilution of JAK2, JAK3 and TYK2: 20 mM 3-(N-morpholine)propanesulfonic acid (MOPS), 1 mM EDTA, 0.01% Brij-35.5% glycerol, 0.1% β-mercaptoethanol, 1 mg/mL BSA; Dilution of JAK1: 20 mM TRIS, 0.2 mM EDTA, 0.1% β-mercaptoethanol, 0.01% Brij-35.5% glycerol. All the compounds were prepared into 100% DMSO solution and the concentration reached final measured concentration of 50 times. The test compound was diluted by a 3-fold concentration gradient, and the final concentration was 9 concentrations from 10 μM to 0.001 μM. The content of DMSO in the detect reaction was 2%. The stock solution of this compound was added into wells as a first component, then the other components were added as the following detailed process. JAK3(h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 500 μM GGEEEEYFELVKKKK, 10 mM magnesium acetate and [γ-33P]-ATP (activities and concentration were customized as required). Mixture of Mg/ATP was added to start reaction, which was stopped by adding 0.5% phosphoric acid after 40 minutes incubation. Then 10 μL of the reaction solution was dispersed on the P30 filter pad, washed with 0.425% phosphoric acid for three times and with methanol for one time within 4 minutes, dried, and scintillate counted. |
| Affinity data for this assay | |
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