| Assay Method Information | |
| | DNA-PK Biochemical Assay |
| Description: | DNA-PK Biochemical AssayDNA-PK kinase reaction solutions of DNA-PK enzymes (Promega, Cat. No. V4106, Lot. No. 0000224016) were prepared in 1× kinase buffer at 2-fold of the final concentration (final concentration: DNA-PK 1 U/μl, Activator 6 μg/ml) of each reagent in the assay. 2.5 μl of kinase solution was added to each well of the 384-well assay plate, which contains 2.5 μl of compounds with serially diluted concentration. Substrate solution was prepared and ATP in 1× kinase reaction buffer at 2-fold of the final concentration of each reagent in the assay. The final concentration: Substrate 0.2 ug/ml and ATP 20 uM. 2.5 μl of substrate solution was added to each well of the assay plate to start reaction. The assay plate was incubated at room temperature for 1 hour. 5 μl reaction mix was transferred to a new 384 well plate. 5 μl of ADP-Glo MAX reagent 1 (Promega, Cat. No. v9102/3, Lot. No. 0000176563) was added to each well of the assay plate to stop the reaction. The plate was equilibrated for 2 hr at room temperature. 10 μl ADP-Glo MAX reagent 2 was added to each well, which was equilibrated for 30 minutes before read on a plate reader (Envision) for luminescence. |
| Affinity data for this assay | |
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