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 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.
Miick
Siobhan M
SM
Gen-Probe Inc., San Diego, CA, USA.
Jalali
Shila
S
Dwyer
Brian P
BP
Havens
John
J
Thomas
Donald
D
Jimenez
Manuel A
MA
Simpson
Mathew T
MT
Zile
Betsy
B
Huss
Karen L
KL
Campbell
Robert M
RM
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
D004791:Enzyme Inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases