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 (, 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:


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.