Abstract
Combinations of multiple fluorescent fusion proteins are commonly generated and used for colocalization studies in live cell imaging but also biochemical analysis of protein–protein interactions by co-immunoprecipitation in vitro. Advanced microscopy techniques like Förster resonance energy transfer through fluorescence lifetime imaging microscopy (FRET/FLIM) nowadays enable the combination of both approaches. This opens up the possibility to perform a location-specific protein–protein interaction analysis in vivo. To this end, the nonradiant energy transfer from a donor to an acceptor fluorophore (FRET) is harnessed to test for close proximity as an indicator for interaction, while the spectromicroscopical measurement of the fluorescence lifetime by FLIM serves as a readout.
Here, we describe FRET/FLIM measurements performed with a Leica TCS SP8/PicoHarp 300 combination to demonstrate the interaction between a RFP-tagged GFP-nanobody and its epitope, GFP, in the cytoplasm of tobacco mesophyll protoplasts.
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Acknowledgments
We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (PI 769/1-2 and the Collaborative Research Centre SFB 1101 “Molecular Encoding of Specificity in Plant Processes”) and of the German Academic Exchange Service (Project 57219822).
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Fäßler, F., Pimpl, P. (2017). In Vivo Interaction Studies by Measuring Förster Resonance Energy Transfer Through Fluorescence Lifetime Imaging Microscopy (FRET/FLIM). In: Jiang, L. (eds) Plant Protein Secretion. Methods in Molecular Biology, vol 1662. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7262-3_14
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DOI: https://doi.org/10.1007/978-1-4939-7262-3_14
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