Add 2.5X Kinase Buffer to the Kinase Strips. Add 100µM ATP to the Substrate Strips. Kinase Working Stocks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2µl Dispense Kinase Working Stocks to the appropriate wells. H G F E D C B A P O N M L K J I 1µl Dispense compound or vehicle to the appropriate wells. Incubate for 60 minutes. Perform ADP-Glo™ Kinase Assay The concentration of each kinase is optimized for optimal ATP conversion, yielding a signal-to-background ratio ≥10. 2µl Dispense ATP/Substrate Working Stocks to the appropriate wells. ATP/Substrate Working Stocks Figure 1. Kinase Selectivity Profiling Systems overview. Kinases are provided at 25X or 50X concentrations in an 8-tube strip, and substrates/ cofactors are provided at 3.3X concentrations in a separate 8-tube strip. One-step dilutions directly in these strips produce sufficient Kinase and ATP/Substrate Working Stocks for 25 kinase reactions in a 384-well plate. After a 1-hour incubation, kinase activity is quantified using the ADP-Glo™ Kinase Assay. The resulting luminescent signal is proportional to ADP concentration and is correlated with kinase activity. A. 150 100 50 JAK3 IC50 TRKA IC50 0 0.01 0.1 1 10 100 1,000 10,000 tofactinib (nM) = 4.2nM = 1910nM 0 0.01 0.1 1 10 100 1,000 10,000 PF-477736 (nM) Figure 2. Dose response profiling of two compounds using the Kinase Selectivity Profiling System: tK-3. The selective JAK3 inhibitor Tofacitinib (Panel A) and nonselective inhibitor PF-477736 (Panel B) were assayed at the indicated concentrations using the Kinase Selectivity Profiling System: TK-3. Kinase assays were assembled using 1µl of inhibitor, 2µl of Kinase Working Stock and 2µL of the corresponding ATP/Substrate Working Stock in a 384-well plate. Assays were incubated for 1 hour at 23°C before kinase activity was quantified using the ADP-Glo™ Kinase Assay. Percent activity of each kinase in the presence of each inhibitor is indicated. Curves were fit to a sigmoidal dose-response (variable slope) equation and IC50 values were calculated. B. AXL1 EPHA1 Fak ITK JAK3 PYK2 SYK TRKA 150 100 50 AXL1 EPHA1 Fak ITK JAK3 PYK2 SYK TRKA enews | Juillet 2015 Percent Enzyme Activity Percent Enzyme Activity 12059MA 12902MA Pagina 6

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