RESEARCH ARTICLE


Development of a HTRF® Kinase Assay for Determination of Syk Activity



Christopher Harbert*, Jeannette Marshall, Sharon Soh, Krista Steger
Cisbio US Inc., 12 Deangelo Dr., Bedford MA 01730, USA


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2008 Bentham Science Publishers Ltd

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.

* Address correspondence to this author at the Cisbio US Inc., 12 Deangelo Dr., Bedford MA 01730, USA; Tel: (888) 963-4567; Fax: (781) 687-1298; E-mail: charbert@cisusinc.com


Abstract

Regulation of protein phosphorylation is a primary cellular signaling mechanism. Many cellular responses to internal and external events are mitigated by protein kinase signaling cascades. Dysfunction of protein kinase activity has been linked to a variety of human pathologies, in the areas of cancer, inflammation, metabolism, cell cycle, apoptosis, as well as cardiovascular, neurodegenerative and autoimmune diseases [1-3]. As such, there is an important need for protein kinase activity detection methodologies for researchers engaged in Drug Discovery. A number of different technologies have been employed for the measurement of protein kinase activity, including radioactive methods, luminescent methods, and fluorescent methods. More recently, Homogeneous Time Resolved Fluorescence technology (HTRF®), based on the principle of time-resolved fluorescent resonance energy transfer (TR-FRET), has been developed and applied for the measurement of protein kinase activity in vitro. This technology note describes the development of an HTRF® assay for detection of Syk enzyme activity in a format consistent with the requirements of High-Throughput Screening (HTS) campaigns currently used in drug discovery.