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

Siobhan M. Miick

Gen-Probe Inc., San Diego, CA; Nanogen, Inc., San Diego, CA

Shila Jalali

Nanogen, Inc., San Diego, CA

Brian P. Dwyer

Nanogen, Inc., San Diego, CA; Invitrogen Corporation, Carlsbad, CA

John Havens

Nanogen, Inc., San Diego, CA

Donald Thomas

Nanogen, Inc., San Diego, CA; ACEA Biosciences, San Diego, CA

Manuel A. Jimenez

Mathew T. Simpson

Nanogen, Inc., San Diego, CA

Betsy Zile

Nanogen, Inc., San Diego, CA; Carmel, IN

Karen L. Huss

Robert M. Campbell

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCaptureTM PKA assay developed uses a positively charged, lissamine-rhodamine–labeled kemptide peptide substrate for the kinase reaction and Nanogen’s ElectroCaptureTM 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 ElectroCaptureTM PKA assay was validated with both known PKA inhibitors and library compounds. The pKiapp results obtained in the ElectroCaptureTM PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.

Key Words: ElectroCaptureTM • protein kinase A • PKA • fluorescence polarization • FP

Journal of Biomolecular Screening, Vol. 10, No. 4, 329-338 (2005)
DOI: 10.1177/1087057104272909
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