title: 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 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 pK(iapp) 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.
authors.affiliation: Gen-Probe Inc., San Diego, CA, USA.
authors.forename: Siobhan M
authors.initials: SM
authors.lastname: Miick
authors.forename: Shila
authors.initials: S
authors.lastname: Jalali
authors.forename: Brian P
authors.initials: BP
authors.lastname: Dwyer
authors.forename: John
authors.initials: J
authors.lastname: Havens
authors.forename: Donald
authors.initials: D
authors.lastname: Thomas
authors.forename: Manuel A
authors.initials: MA
authors.lastname: Jimenez
authors.forename: Mathew T
authors.initials: MT
authors.lastname: Simpson
authors.forename: Betsy
authors.initials: B
authors.lastname: Zile
authors.forename: Karen L
authors.initials: KL
authors.lastname: Huss
authors.forename: Robert M
authors.initials: RM
authors.lastname: Campbell
mesh_terms: D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005374:Filtration; D005454:Fluorescence Polarization / Q000379:methods*; D007700:Kinetics; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases