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 tofactinib (nM) 1,000 10,000 = 4.2nM = 1910nM 0 0.01 0.1 1 10 100 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. 1,000 10,000 B. AXL1 EPHA1 Fak ITK JAK3 PYK2 SYK TRKA 150 100 50 AXL1 EPHA1 Fak ITK JAK3 PYK2 SYK TRKA enews | April 2015 Percent Enzyme Activity Percent Enzyme Activity 12059MA 12902MA Pagina 6
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Promega Benelux - Monthly online Magazine - April 2015 edition Lees publicatie 100Home