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Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission
- Source :
- Journal of visualized experiments : JoVE. (170)
- Publication Year :
- 2021
-
Abstract
- Forster 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.
- Subjects :
- Microscopy
Quenching (fluorescence)
Fluorophore
General Immunology and Microbiology
Absorption spectroscopy
Cell Survival
General Chemical Engineering
General Neuroscience
Proteins
Fluorescence
Acceptor
General Biochemistry, Genetics and Molecular Biology
Rats
chemistry.chemical_compound
Förster resonance energy transfer
chemistry
Live cell imaging
Excited state
biological sciences
Biophysics
Fluorescence Resonance Energy Transfer
Animals
Subjects
Details
- ISSN :
- 1940087X
- Issue :
- 170
- Database :
- OpenAIRE
- Journal :
- Journal of visualized experiments : JoVE
- Accession number :
- edsair.doi.dedup.....4af7861392895e0ebe27c097f50f28f4