Optimization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for Varioskan®

Marika Raitio, Thermo Fisher Scientific, Finland

Abstract

Fluorescence resonance energy transfer (FRET) is a technique used to measure distance-dependent interactions on the scale of approximately 15-100 Å. It is based on usage of two different labels called donor and acceptor. The donor has to be fluorescent and the acceptor able to receive donor energy (Figure 1). The donor is excited in the process and if the acceptor is in close proximity the excited state energy can be transferred. This leads to a reduction in the donor fluorescence intensity and excited state lifetime, and an increase in the acceptors emission intensity. Therefore there are several parameters to measure in order to detect energy transfer: change in donor intensity, lifetime and acceptor intensity. FRET includes no intermediate photon but the donor and acceptor are coupled by dipole-dipole interaction. In this particular assay the acceptor intensity is measured