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Methods Mol Biol. 2009;572:161-76. doi: 10.1007/978-1-60761-244-5_10.

Fluorescence polarization and time-resolved fluorescence resonance energy transfer techniques for PI3K assays.

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  • 1Reaction Biology Corporation, Malvern, PA, USA.

Abstract

Fluorescence-based biochemical assays are sensitive and convenient to use; therefore, they are widely employed for enzyme assays and molecular interaction studies. However, when this method is applied for screening of a compound library for drug discovery, high fluorescence compounds, which usually exist in large numbers in chemical libraries, are problematic. Fluorescence Polarization (FP) and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays are less affected by compound fluorescence and suitable for large-scale high-throughput screening (HTS). In this section, we describe homogenous FP and TR-FRET methods for PI3-kinase (PI3K), a family of lipid kinases that is "difficult-to-do-HTS" since traditional radioisotope assays are hard to apply to HTS format. The application of FP and TR-FRET techniques for PI3K HTS will be described and advantages and disadvantages of these assays will be discussed.

PMID:
20694691
[PubMed - indexed for MEDLINE]
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