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

摘要

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.