Development of a Microplate-Based, Electrophoretic Fluorescent Protein Kinase A Assay: Comparison with Filter-Binding and Fluorescence Polarization Assay Formats

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture™ PKA assay developed uses a positively charged, lissamine-rhodamine–labeled kemptide peptide substrate for the kinase reaction and Nanogen’s ElectroCapture™ HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture™ PKA assay was validated with both known PKA inhibitors and library compounds. The pKiapp results obtained in the ElectroCapture™ PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats. (Journal of Biomolecular Screening 2005:329-338)