Fluorescence Resonance Energy Transfer (FRET)
Fluorescence Resonance Energy Transfer (FRET)
Fluorescence Resonance Energy Transfer (FRET)
Principle
As an efficient optical "molecular ruler", fluorescence resonance energy transfer (FRET) is a physical method to detect intermolecular interactions, which means that when two fluorescent chromophores are in close enough proximity, the donor molecule absorbs a certain frequency of photons and is excited to a higher electron-energy state, and the electron returns to the ground state through a dipole interaction. energy state, and the energy transfer to the neighboring acceptor molecule is achieved through dipole interactions before that electron returns to the ground state, i.e., energy resonance transfer occurs.
The degree of FRET is closely related to the spatial distance of the group QC, and FRET generally occurs at 7-10 nm; with the extension of the distance, FRET is significantly weakened. The efficiency of FRET between groups can be reflected by E = 1/1 + (R/R0)exp6, where R denotes the distance between groups and R0 denotes the Fuchsian radius, which depends on the degree of overlap between the emission spectrum of the fluorescent group and the excitation spectrum of the quenched group, as well as on the relative orientation of the dipoles of the group energy transfer.
Operation method
Live cell FRET analysis
Principle
As an efficient optical "molecular ruler", fluorescence resonance energy transfer (FRET) is a physical method to detect intermolecular interactions, which means that when two fluorescent chromophores are in close enough proximity, the donor molecule absorbs a certain frequency of photons and is excited to a higher electron-energy state, and the energy is transferred to the neighboring acceptor molecule through dipole interaction before the electron returns to the ground state. When two fluorescent chromophores are close enough, the donor molecule is excited to a higher electronic state after absorbing a certain frequency photon, and the energy is transferred to the neighboring acceptor molecule through dipole interaction before the electron returns to the ground state, i.e., the energy resonance transfer occurs.The degree of FRET is closely correlated with the spatial distance of the chromophores, and the FRET generally occurs when the distance is 7-10 nm; as the distance is lengthened, the FRET is significantly weakened. The efficiency of FRET between groups can be reflected by E = 1/1 + (R/R0)exp6, where R denotes the distance between groups and R0 denotes the Fuchsian radius, depending on the degree of overlap between the emission spectrum of the fluorescent group and the excitation spectrum of the quenched group, as well as the relative orientations of the dipoles of the group energy transfer.
Materials and Instruments
Enzyme marker, two fluorescent chromophores, cell line, plasmid vector Move The fluorescent substance CFP (donor)-YFP (acceptor) was used as an example to detect the interaction of AB proteins in cells. 1, Cell transfection: Construct the plasmid vectors CFP-A and YFP-B, couple the detected AB proteins to the fluorescent proteins CFP and YFP, respectively, and co-transfect the two plasmids into the cultured cells; 2. 24 hours after transfection, remove the culture medium and fix the cells with 4% paraformaldehyde for 15 minutes, and wash twice with PBS; 3. FRET image acquisition: The excitation wavelengths of FRET pairs were determined, and the blue light was tuned to detect the maximum and minimum donor and acceptor signals, respectively, where the wavelengths corresponding to the maximum donor and minimum acceptor signals were used to collect FRET signals from the dual-expression cells. The excitation light wavelength was adjusted to obtain the maximum CFP signal and the minimum YFP signal, the donor and acceptor images were acquired, and the FRET signal was collected. Repeat the FRET assay 3 times for each cell, and complete the FRET assay for at least 6 cells for each transfection. 4. FRET data processing: For image analysis and coefficient calculation, each image should be subtracted from the background, and the spectral crosstalk signals of DSBT and ASBT should be removed from the FRET signals; in addition, the FRET signals of the acceptor pathway need to be corrected for the change of the spectral sensitivity of the donor and acceptor, and corrected for the autofluorescence and optical noise; meanwhile, pixel-matching correction should be carried out for the double-calibrated cells by using correction coefficients. Caveat 1. the spatial distance between the donor and acceptor fluorophores should be close enough to be ＜10 nm;2. the emission spectrum of the donor and the reception spectrum of the acceptor have to overlap considerably;3. the fluorescent chromophores of the energy donor and the energy acceptor must be arranged in an appropriate manner. For more product details, please visit Aladdin Scientific website.