Title: An enzyme-linked immunosorbent assay for protein kinase D activity using phosphorylation site-specific antibodies
Authors: Rykx, An *
Vancauwenbergh, Sadia *
De Kimpe, Line *
Janssens, Katrien
Vandoninck, Sandy
Waelkens, Etienne
Vandenheede, Jackie
Van Lint, Johan # ×
Issue Date: Oct-2007
Series Title: Assay and drug development technologies vol:5 issue:5 pages:637-643
Abstract: The protein kinase D (PKD) family is a novel group of kinases that are involved in the regulation of cell proliferation and apoptosis, and several other physiological processes. Hence, these enzymes are attractive targets for pharmacological intervention, but no specific PKD inhibitors are known. With this in mind, we have developed a high-throughput, non-radioactive enzyme-linked immunosorbent assay (ELISA) method to monitor the PKD activity with myelin basic protein (MBP) as substrate. We determined that MBP is phosphorylated by PKD on Ser-160 and that this phosphorylation can be quantified in ELISAs, by the use of phosphorylation site-specific antibodies. Antibodies were developed that are highly specific for the MBP peptide sequence surrounding the phosphorylated Ser-160. We show that our high-throughput kinase assay is useful not only for determining the cellular PKD activity but also to screen for PKD-inhibitory compounds. Our ELISA has advantages over the current radioisotope kinase assay in terms of simplicity and environmental safety.
ISSN: 1540-658X
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Biochemistry Section (Medicine) (-)
Laboratory of Protein Phosphorylation and Proteomics
Laboratory of Phosphoproteomics (-)
* (joint) first author
× corresponding author
# (joint) last author

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