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| | WNK1 HTRF Assay |
| Description: | For hit validation, eight compound replicates were made from the original stock dilution plates. The Cybi-WellTM dispenser was used to prepare 1.5 μL of compound dots in the intermediate dilution plates, which were then transferred to the assay plates. For validation at 100 μM ATP, the reactions were carried out and quenched and products detected as described above for screening. For validation at 1 mM ATP, the compound transfer and reagent preparation followed the same standard assay protocol except for using 10× higher ATP in the substrate mixture. The reactions were incubated for 1.5 h at RT, at which time point the signal generation is still in the linear range. A total of 6 μL of the stop and detection reagent was added and incubated at RT for at least 2 h before reading on an Envision Multilabel reader (PerkinElmer). All compounds were tested in duplicate as an eight-point, half-log dilution series from 0.025 to 80 μM. |
| Affinity data for this assay | |
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