| Assay Method Information | |
| | ADP-Glo Kinase Assay |
| Description: | Experimental materials: JAK1 kinase (Invitrogen, PV4744), ATP (Promega, V915B), ADP-Glo Kinase Assay (Promega, V9101), IRS1 (Signalchem, 140-58-1000). Sample preparation: The compounds of the present invention and the control product were dissolved in DMSO solvent respectively to formulate into 10 mM mother liquor. The final reaction maximum concentration of the compound was 10 μM, 3-fold dilution, 10 concentration gradients, and duplicate wells for each concentration gradient.Experimental process: 0.1 μL of the compound to be tested was transferred into a 384-well reaction plate (PE, 6007290) via Echo and centrifuged at 1000 rpm/min for 1 min. 5 μL of JAK1 kinase (final concentration of 4 nM) was transferred into the 384-well reaction plate, which was then centrifuged at 1000 rpm/min for 1 min, and incubated at 25° C. for 15 min. 5 μL of substrate mixture (1 mM ATP, IRS1 0.05 mg/ml, kinase buffer solution) was transferred into the 384-well reaction plate, which was then centrifuged at 1000 rpm/min for 1 min, and incubated at 25° C. for 60 min. 10 μL of ADP-Glo was transferred into the 384-well reaction plate, which was then centrifuged at 1000 rpm/min for 1 min, and incubated at 25° C. for 40 min. 20 μL of test solution was transferred into the 384-well reaction plate, which was then centrifuged at 1000 rpm/min for 1 min and incubated at 25° C. for 40 min. RLU (Relative luminescence unit) signal was read by using an Envision multifunctional plate reader. |
| Affinity data for this assay | |
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