Detecting Protein Interactions with Red-Shifted FRET
Author Information
Author(s): Goedhart Joachim, Vermeer Joop E. M., Adjobo-Hermans Merel J. W., van Weeren Laura, Gadella Theodorus W. J. Jr.
Primary Institution: University of Amsterdam
Hypothesis
Red-shifted FRET couples provide superior alternatives to the CFP/YFP couple for the detection of protein-protein interactions in single living cells.
Conclusion
Red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells.
Supporting Evidence
- The red-shifted FRET pairs displayed a relatively high FRET efficiency.
- The most favorable orange-red FRET pair was mKO-mCherry.
- Red-shifted FRET pairs showed a threefold higher lifetime contrast than CFP-YFP.
Takeaway
This study shows that using special red fluorescent proteins can help scientists see how proteins interact in living cells better than older methods.
Methodology
The study used fluorescence lifetime imaging microscopy (FLIM) and acceptor photobleaching to measure FRET efficiencies of various fluorescent protein pairs in living cells.
Limitations
The FRET efficiency in tandem constructs may not regularly depend on the length of the linker, and the results cannot always be directly compared to FRET efficiencies obtained in other studies.
Participant Demographics
HeLa cells were used for the experiments.
Digital Object Identifier (DOI)
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