A Filter-Based, 96-Well Microtiter Plate Calcineurin Phosphatase Assay

A rapid, sensitive, and specific filter-based assay for calcineurin phosphatase that utilizes a 96-well microtiter plate format throughout is described. In this assay, either purified calcineurin phosphatase or a crude cellular extract containing calcineurin phosphatase is incubated with a 33P-labeled phosphopeptide substrate in a 96-well microtiter plate. Subsequent to quenching the dephosphorylation reaction, the incubation mixture is transferred to a 96-well Immobilon-P microtiter filtration plate affixed to a vacuum manifold. The phosphorylated peptide remains bound to the hydrophobic filter. The filtrate, containing the released 33P-labeled phosphate, is collected in a 96-well microtiter plate and is measured by liquid scintillation counting.