| Assay Method Information | |
| | In vitro MBP Phosphorylation Assay (MBP) |
| Description: | Each well of the MBP-coated ScintiPlates held 100 ul of a solution containing 20 mM HEPES 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 uM ATP (WNK1, WNK3 and WNK4) or 2 uM ATP (WNK2), 1 uCi [gamma-33P]ATP (WNK1, WNK2, WNK4) or 0.25 uCi [gamma-33P]ATP (WNK3), WNK kinase enzyme (5 nM WNK1, 10 nM WNK2, 5 nM WNK3 or 10 nM WNK4) and compound at the desired concentration. The plate was sealed with a clear adhesive cover and mixed for 20 s at 800 r.p.m. on a bench top plate shaker. The plate was then placed in a 25 °C shaking incubator at 175 r.p.m for 3 h (WNK1 and WNK4), 2 h (WNK3) or 1 h (WNK2). The kinase reaction was stopped by the addition of 50 ul of 45 mM EDTA, 0.01% Tween-20, and 20 mM HEPES, pH 7.3. The content of each well was then aspirated, and the well was washed three times with 300 ul of 150 mM NaCl, 0.02% Tween-20, and 50 mM Tris-HCl pH 7.4. Incorporation of 33P into the bound MBP substrate was measured using a MicroBeta TriLux LSC and Luminescence plate counter. |
| Affinity data for this assay | |
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