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| | Microfluidic Mobility-Shift Assay |
| Description: | Kinase activity was determined employing a microfluidic mobility-shift assay on a Caliper DeskTop Profiler (Caliper Life Sciences, PerkinElmer). The protein was incubated for 2 h at ambient temperature in a 384-well assay plate (Corning LV, nonbinding surface) in 20 uL of buffer (20 mM 3-(Nmorpholino) propanesulfonic acid, at pH 7.0, 300 mM sodium chloride, 1 mM dithiothreitol, 0.05% L-31, 10 uM fluorescein isothiocyanate-kemptide, 990 uM kemptide, 1 mM ATP, 10 mM magnesium chloride) and various concentrations of cGMP, 8-BrcGMP, 8-pCPT-cGMP, and PET-cGMP (Biolog life science), respectively. Reaction mixtures without cyclic nucleotide were used as controls. A ProfilerPro LabChip (4-sipper mode; PerkinElmer) was used for electrophoretic separation of the substrate and product. |
| Affinity data for this assay | |
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