Optimization of LanthaScreen Terbium-Fluorescein TR-FRET Assay with Varioskan® Spectral Scanning Time-Resolved Fluorometer

Hanna Granö, Jorma Lampinen, Veera Välisalo and Marika J. Raitio, Thermo Fisher Scientific, Vantaa, Finland

Abstract

Time-resolved fluorescence energy transfer (TR-FRET) is a commonly used variation of fluorescence energy transfer (FRET), and it uses time-resolved fluorescence label as FRET donor. Compared to fluorescence intensity based FRET, it has a benefit of lacking biological background fluorescence because of the time dependent phenomena. In this work, measurement parameters for LanthascreenTM TR-FRET assay were optimized using Varioskan® spectral scanning microtiter plate fluorometer. Excitation and emission spectra of free donor and TR-FRET combination were measured using time-resolved spectral scanning and resulting spectra were analyzed to determine best possible resolution between donor and TR-FRET emissions. Additionally, time-resolved fluorescence signal decay curves over the time from the donor and TR-FRET complex were measured to determine optimal measurement window for the TR-FRET assay.