Fluorescence Resonance Energy Transfer (FRET)
Protein-protein interactions are critical for cellular function and a major target class for drug discovery, yet they are difficult to investigate. FRET (Förster or Fluorescence Resonance Energy Transfer) is the non-radiative transfer of energy from an excited fluorophore (donor) to another fluorophore (acceptor). FRET only occurs when the molecules concerned are in very close proximity. This makes it a valuable technique for studying interactions between molecules, such as proteins, in solution or in cells. Exciting the donor and then monitoring the relative donor and acceptor emissions, either sequentially or simultaneously, makes it possible to determine when FRET has occurred.
Detection of FRET can be used to quantify when and where two or more biomolecules interact. As the energy transfer occurs only over distances between 1-10 nm, the distance between interacting proteins can be measured with a precision beyond the optical resolution of the light microscope (~0.25 µm). As a result, FRET is a useful tool to quantify molecular dynamics, such as protein-protein interactions, protein-DNA interactions, and conformational changes of molecules in living cells.
FRET can be either intramolecular or intermolecular. Intramolecular show changes of FRET intensity as a result of conformation changes or substrate cleavage (e.g. MMPSense). Intermolecular FRET requires two fluorophores to move in very close proximity.
A common combination of fluorophores for studying protein-protein interactions in cells of interest is cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor-acceptor pair.
When CFP is excited and the two molecules are within 10 nm of each other, energy may be transferred from the excited CFP to the YFP causing the YFP to emit yellow light, which is then detected independently of the blue light that excited CFP.
PerkinElmer High-Content Systems for FRET Assays
The Opera Phenix™ high-content screening system offers many benefits for FRET assays. For working with CFP/YFP type fluorophores, Opera Phenix™ can be configured with a 425 nm laser for optimal excitation of the CFP donor. Furthermore, donor and sensitized acceptor emission can be recorded simultaneously when working with a two or four camera system. The four camera set-up enables you to record a full FRET experiment including the two FRET emissions, a nuclear marker and an additional marker of interest in a single exposure, while the microlens enhanced confocal Synchrony Optics™ minimize bleaching and photodamage to the cells. Water immersion objectives and state-of-the-art sCMOS cameras ensure high sensitivity.
With its sensitive imaging and dedicated analysis tools for ratiometric imaging, robust results are also easy to obtain with the Operetta CLS™ high-content analysis system.
Harmony high-content imaging and analysis software offers dedicated analysis and visualization tools for ratiometric imaging.