Development of a microplate-based, electrophoretic fluorescent protein kinase A assay: Comparison with filter-binding and fluorescence polarization assay formats

Abstract

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 ElectroCaprure™ 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 pKi app 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. © 2005 The Society for Biomolecular Screening.