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Förster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.
Förster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.
Microscopy-based analysis of Förster Resonance Energy Transfer (FRET) permits assessment of interactions between proteins in live cells. It provides spatial and temporal information, including information on where in the cell and in which subcellular compartment the interaction takes place and if this interaction changes over time.
Theodor Förster laid the theoretical foundation of FRET in 19481. FRET is a radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance of the molecules and the relative orientation of their transition dipoles as well as the overla....
1. Plasmid construction
Figure 1 shows the images obtained in the donor channel, channel 1 (488, 505-530 nm), the transfer channel, channel 2 (488, >585 nm), and the acceptor channel, channel 3 (561, >585 nm), respectively. Representative images of cells expressing GFP only, Cherry only, co-expressing GFP and Cherry, and expressing the GFP-Cherry fusion protein. The mean cellular FRET efficiencies calculated in NRK cells expressing GFP-Cherry fusion protein (positive co.......
The presented protocol details the use of the genetically coupled one-to-one fluorescent protein calibration probe for quantifying FRET using the detection of sensitized emission of the acceptor and quenching of the donor molecule by confocal microscopy. This method can be applied to assess protein interactions in the physiological context of the living cell in different subcellular compartments. Spatial resolution can be further improved by applying the presented algorithm to calculate FRET efficiencies in each pixel of.......
We would like to thank the Neuroscience Imaging Service at Stanford University School of Medicine for providing equipment and space for this project. This research was supported by intramural funding of the Stanford Cancer Institute and the Gynecologic Oncology Division Stanford as well as GINOP-2.3.2-15-2016-00026, GINOP-2.3.3-15-2016-00030, NN129371, ANN135107 from the National Research, Development and Innovation Office, Hungary.
....Name | Company | Catalog Number | Comments |
0.5% Trypsin-EDTA without phenol red (10x) | Thermo Fisher Scientific | 15400054 | |
Clontech mCherry N1 vector | Addgene | 3553 | |
DMEM without phenol red | Thermo Fisher Scientific | 11054020 | |
Fugene 6 | Promega | E2691 | |
HEPES | Thermo Fisher Scientific | 15630080 | |
LabTek 8-well chambers #1.0 | Thermo Fisher Scientific | 12565470 | |
L-Glutamine (200 mM) | Thermo Fisher Scientific | 25030081 |
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