Fret Read Mode - Molecular Devices SpectraMax Paradigm User Manual

SpectraMax Paradigm Multi-Mode Detection Platform User Guide

FRET Read Mode

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between
the electronic excited states of two dye molecules in which excitation is transferred from a
donor molecule to an acceptor molecule without emission of a photon.
FRET relies on the distance-dependent transfer of energy from a donor molecule to an
acceptor molecule. Due to its sensitivity to distance, FRET has been used to investigate
molecular interactions. FRET is the radiationless transmission of energy from a donor
molecule to an acceptor molecule. The donor molecule is the dye or chromophore that
initially absorbs the energy and the acceptor is the chromophore to which the energy is
subsequently transferred. This resonance interaction occurs over greater than interatomic
distances, without conversion to thermal energy, and without a molecular collision. The
transfer of energy leads to a reduction in the fluorescence intensity and excited state lifetime
of the donor, and an increase in the emission intensity of the acceptor. A pair of molecules
that interact in such a manner that FRET occurs is often referred to as a donor/acceptor pair.
While there are many factors that influence FRET, the primary conditions that need to be
met for FRET to occur are relatively few:
The donor and acceptor molecules must be in close proximity to each other.
The absorption or excitation spectrum of the acceptor must overlap the fluorescence
emission spectrum of the donor.
The degree to which they overlap is referred to as the spectral overlap integral (J).
The donor and acceptor transition must be approximately parallel.
You can use the Protocol Manager in the SoftMax Pro Software to quickly find and open a
predefined protocol.
More protocols and updated protocols can be downloaded from the Knowledge Base on the
Molecular Devices support web site (www.moleculardevices.com/support) or from the
protocol sharing web site (www.softmaxpro.org).
The following detection cartridge has FRET read mode capability:
Cisbio HTRF Detection Cartridge, see page 110
44 5014038 E