( P J. Turner ...)Homogeneous, chemiluminescent kinase assay kits for assessing the functional activity of a broad range of serine-threonine and tyrosine kinases

J. Turner
GE Healthcare, The Maynard Centre, Cardiff, UK

Competitive binding assays have been developed for measuring the functional activity of a range of serine-threonine and tyrosine kinases. Based on enzyme fragment complementation and the subsequent hydrolysis of chemiluminescent substrate, HitHunter™ Kinase Assay Kits generate a signal that can be read either on high-throughput imaging platforms or on standard microplate readers.

Introduction
Human protein kinases are involved in numerous and diverse cellular processes such as signal transduction and cell cycle control. The precise control of protein phosphorylation is fundamental to normal cellular behaviour and uncontrolled signaling by kinases has been implicated in disease states such as cancer, athersclerosis, psoriasis, and in inflammatory responses such as septic shock. Inhibitors that block the activity of protein kinases and phosphatases are therefore potentially useful for the development of therapeutic agents.

HitHunter Kinase Assay Kits are competitive binding assays utilizing enzyme fragment complementation (EFC) for the sensitive and simple measurement of a range of biologically important targets. Based on proprietary kinase antibody/substrate pairs, they provide a sensitive and homogeneous method for measuring the activity of a broad range of serine-threonine and tyrosine kinases covering 80% of the kinome.

HitHunter Kinase Assay principle
EFC technology is based on the recombination of two inactive β-galactosidase (β-gal) fragments; complementation of the enzyme donor (ED) fragment and the enzyme acceptor (EA) fragment forms the active tetrameric β-gal (Fig 1). Subsequent hydrolysis of luminescent substrate generates a signal that can be easily read on standard microplate reader
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