Fluorescence Resonance Energy Transfer (FRET) Service

What is Fluorescence Resonance Energy Transfer?

Nowadays, fluorescence resonance energy transfer (FRET) is increasingly used in biomedical research. FRET relies on the distance-dependent transfer of energy from donor molecules to acceptor molecules, so it is widely used to study molecular interactions. The donor molecule is the dye or chromophore that initially absorbs energy, and the acceptor is the chromophore that subsequently transfers energy. This resonance interaction is not converted into heat energy, nor is there any molecular collision. For FRET to occur, the following conditions must be met: 1. The donor and acceptor fluorophores must be close to each other; 2. The emission spectrum of the donor fluorophore must overlap with the absorption spectrum of the acceptor fluorophore; 3. The energy transfer from the donor fluorophore to the acceptor fluorophore occurs via intermolecular dipole-dipole coupling.

FRET can detect the proximity between two molecules within a few nanometers, so when the distance-related interaction is close enough, FRET can provide information about the interaction between labeled molecules, such as the binding of ligand and receptor. FRET is sensitive, simple operation, and easily automated, so it is also very suitable for high-throughput screening. But FRET relies heavily on the use of high-quality labeling reagents.

Different measures, such as FRET efficiency (E), which indicates the percentage of energy transferred from the donor to the acceptor, can be used to measure FRET efficiency. FRET efficiency can be determined using variations in lifespan data, acceptor fluorescence intensity, or both. Quantitative data from FRET data is extracted using various mathematical models and techniques.

To establish the specificity of the reported FRET signal, control experiments in FRET are also crucial. Positive controls can contain samples with known FRET pairings or interactions, whereas negative controls use materials without the acceptor or with fluorophores that do not interact. These check studies provide a baseline and confirm the precision of the FRET data.

To get over restrictions and increase the functionality of FRET, numerous advanced FRET approaches have been created in addition to regular FRET. These include fluorescence lifetime-based FRET, TR-FRET, multiplexed FRET, single-molecule FRET (smFRET), and fluorescence cross-correlation spectroscopy (FCCS). Higher sensitivity, better spatial resolution, and the capacity to observe dynamic molecular interactions are all provided by these approaches.

Figure 1. Schematic representations of cell surface FRET detection technologies (Fernández-Dueñas, V.; et al.;2012)

FRET Platform at Creative Proteomics

Our advanced FRET technology platform and well-trained technicians can provide guarantee for customers' projects. We provide a wide range of fluorescently labeled antibodies, proteins, and dyes, covering the spectrum from ultraviolet to far-infrared to meet all your experimental needs. Our best quality FRET microscope imaging services include world-class equipment, flexible and diverse choices of fluorophore pairs, and professional image analysis tools. And our one-stop service covers every step of the project, including fluorophore fusion protein construction, cell preparation, fluorescence imaging, image processing and data analysis, etc.

Figure 2. FRET imaging microscopy experiment (Truong, K.; Ikura. M. 2001)

Creative Proteomics also offers TR-FRET services. A TR-FRET (Time-Resolved Förster Resonance Energy Transfer) platform is a combination of time-resolved fluorescence (TRF) and Förster resonance energy transfer (FRET) techniques for studying molecular interactions and assays. It utilizes the principles of both TRF and FRET to provide enhanced sensitivity and improved signal-to-noise ratio in fluorescence-based assays.

In a TR-FRET platform, the donor fluorophore is typically a long-lived lanthanide complex, such as europium or terbium, which emits fluorescence with a long decay time. The acceptor fluorophore is usually a shorter-lived dye that undergoes FRET when in close proximity to the donor.

Workflow of the TR-FRET platform:

• Donor excitation: The sample is excited with a suitable light source (often a laser or a flashlamp) at a wavelength that specifically excites the donor fluorophore.
• Donor emission: The excited donor emits fluorescence, which has a long decay time.
• Delayed emission detection: A time-gated detection system is employed to collect the delayed emission from the donor fluorophore, effectively eliminating background fluorescence and scattered excitation light.
• Energy transfer: If the acceptor fluorophore is in close proximity to the donor, energy transfer via FRET occurs. The acceptor absorbs the energy from the donor and subsequently emits fluorescence.
• Signal detection: The fluorescence emission from the acceptor is detected using appropriate filters and detectors. The intensity of acceptor fluorescence is measured to determine the extent of FRET, which can be correlated with the molecular interactions or assay readout.

TR-FRET platforms have several advantages:

• Reduced background noise: The delayed emission detection of TRF allows for efficient rejection of short-lived background fluorescence, resulting in a higher signal-to-noise ratio.
• Extended measurement window: TRF utilizes long-lived fluorophores with microsecond to millisecond lifetimes, enabling extended measurement windows and flexibility in assay development.
• Sensitivity: The combination of TRF and FRET enhances the sensitivity of the assay, making it suitable for detecting low concentrations of analytes or studying weak molecular interactions.

Other advanced FRET techniques:

Multiplexed FRET: This technique involves the simultaneous detection of multiple FRET pairs, enabling the study of multiple molecular interactions or events within the same sample.

Single-Molecule FRET (smFRET): smFRET allows the investigation of individual molecules, providing insights into dynamic conformational changes, interactions, and heterogeneity within a population of molecules.

Fluorescence Cross-Correlation Spectroscopy (FCCS): FCCS measures the coincidence of fluorescence signals from two interacting molecules, providing information about their diffusion rates, stoichiometry, and binding kinetics.

Fluorescence Lifetime-Based FRET: This approach relies on measuring the fluorescence lifetimes of donor and acceptor fluorophores to quantify FRET efficiency. It is particularly useful for investigating dynamic interactions and interactions occurring at different timescales.

Customers can choose different technology platforms according to project requirements, or contact us directly for consultation, and our expert team will provide you with customized experimental procedures.

Highlights of FRET Platform

• Real-time data collection
• Can capture instantaneous interactions
• Study protein state in vivo
• High accuracy and high sensitivity
• Suitable for different tissues and cell types
• Minimal photo-bleaching and less photo-damage

Applications of FRET Service

• Study on the interactions between intracellular molecules
• Study on the interactions between cell membrane receptors
• Study on localization modification of membrane protein
• Apoptosis research
• Detect changes in enzyme activity
• Receptor-ligand binding studies
• Protein interaction analysis
• Drug discovery

Creative Proteomics is an international biotechnology company dedicated to research in molecular interactions and other related fields. The excellent fluorescence resonance energy transfer technology platform we have built can provide customers with high-quality, one-stop research services, and help customers study various interactions more effectively.