| Assay Method Information | |
| | WNK1 Mobility Shift Assay |
| Description: | A mixture of fluorescein labeled OSR1 peptide substrate (Toray Research Center, Inc.) and ATP was prepared with final concentrations of 10 and 25 μM, respectively, in 7 μL of reaction buffer [20 mM HEPES Na (pH 7.5), 1 mM MnCl2, 0.01% Tween 20, and 2 mM DTT]. Compounds at final concentrations from 0.003 to 100 μM in 1 μL were added to the peptide/ATP mixture at a final DMSO concentration of 10%. The reaction was initiated by the addition of 2 μL of GST-WNK1 (1−491; Carna Biosciences) that was 86% pure at a final concentration of 25 nM in a 384-well plate. After incubation for 3 h at 25 °C, where 15% conversion of the substrate to the product was observed, 10 μL of a stop buffer containing 0.2% coating-3 reagent with 2 mM EDTA was added. For 800 μM ATP, the WNK1 assay was performed for 50 min at 25 °C, where 15% conversion of the substrate to the product was observed. Aliquots from each well were transferred onto a four-sipper chip on a Labchip3 |
| Affinity data for this assay | |
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