Current status of HTRF^® technology in kinase assays

Abstract

Background: Homogeneous time-resolved fluorescence (HTRF) is a fluorescence resonance energy transfer-based technology used to measure bimolecular interactions. It has been applied successfully to kinase assays and has become an important tool in kinase drug discovery. Objective: This article reviews the current status of HTRF technology in biochemical and cellular kinase assays. Methods: Recent literature and meeting reports on HTRF kinase assays are reviewed, and their principles, advantages and drawbacks, current status and the potential applications in kinase drug discovery are discussed. Results/conclusion: HTRF kinase assays are homogeneous, robust, sensitive, easy to miniaturize and high-throughput. This assay format is versatile, as both peptide and protein substrates can be used, and high ATP concentrations are tolerated, which enables the assay to be performed under conditions mimicking the physiological environment. HTRF kinase assays have been applied to both high-throughput screening and compound mechanistic studies. Besides protein kinases, the technology has now been expanded into the lipid kinase family. Furthermore, the utility of HTRF technology in cellular assays is emerging. HTRF kinase assays are a great addition to the toolbox for kinase drug discovery.

Keywords::

• assay
• europium-cryptate
• FRET
• HTRF
• kinase
• phospho-specific antibodies
• SAXL

Acknowledgements

I thank C Quinn for editing the manuscript and for discussions. I also thank S Soh and C Balagtas for proof-reading the manuscript and for feedback and suggestions.

Notes