| Assay Method Information | |
| | WNK In Vitro Radiometric Assays |
| Description: | The assay utilized 5 to 10 nM of WNK1−4 protein compared to 25 nM used for mobility shift assay, enabling a more accurate comparison of selectivity among potent inhibitors. Assays measured the incorporation of 33P from [γ-33P]ATP into myelin basic protein (MBP) coated in the wells of 96-well ScintiPlates (PerkinElmer). Each well of the MBP-coated ScintiPlates held 100 μL of a solution containing 20 mM HEPES at pH 7.3, 5 mM MnCl2 (WNK1 and WNK4) or 3 mM MnCl2 (WNK2 and WNK3), 0.01% Tween-20 (WNK1, WNK3, and WNK4) or 0.02% Tween-20 (WNK2), 1 mM TCEP, 2% DMSO, 1 μM ATP (WNK1, WNK3, and WNK4) or 2 μM ATP (WNK2), 1 μCi [γ -33P]ATP (WNK1, WNK2, WNK4) or 0.25 μCi [γ-33P]ATP (WNK3), WNK kinase enzyme (5 nM WNK1, 10 nM WNK2, 5 nM WNK3, or 10 nM WNK4), and the compound at the desired concentration. The plate was sealed and mixed for 20 s at 800 rpm on a benchtop plate shaker. The plate was then placed in a 25 °C shaking incubator at 175 rpm. Reactio |
| Affinity data for this assay | |
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