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P2004
Ru TR-FRET Assays  Evaluation of Two Acceptor Dyes & Comparison with Eu TR-FRET Assays
Presenter Lin Gao, Hoffmann-La Roche, USA
Additional Authors: Dave Solis, Janna Holmgren, and Kuo-Sen Huang
Time-resolved fluorescence resonance energy transfer (TR-FRET) assays have been widely used in drug discovery. This assay technology exploits the unique fluorescence properties of lanthanide chelates  long fluorescence decay (up to millisecond) allows time-delayed signal detection to eliminate backgrounds and a large Stokes shift that minimizes crosstalk, resulting in high signal-to-background ratio. The most commonly used lanthanide chelates are europium (Eu) and terbium (Tb); and their respective acceptors are allophycocyanin (APC) and fluorescein. Recently, a TR-FRET assay utilizing ruthenium (Ru) complex as the donor and Alexa700 as the acceptor has been developed and successfully used for screening inhibitors of kinases, nuclear hormone receptors and for detection of cAMP in GPCR cell signaling. To further explore the Ru TR-FRET technology, we evaluated a new acceptor dye (Quasar® 683 WS) for Ru in several different assays. Our results suggested that compared to Alexa700, Q683WS increased assay sensitivity. Furthermore, our studies showed that Ru TR-FRET had similar assay sensitivity and quality (Z’ factor) as Eu TR-FRET.