Chapter title |
In Vivo Interaction Studies by Measuring Förster Resonance Energy Transfer Through Fluorescence Lifetime Imaging Microscopy (FRET/FLIM)
|
---|---|
Chapter number | 14 |
Book title |
Plant Protein Secretion
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7262-3_14 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7261-6, 978-1-4939-7262-3
|
Authors |
Florian Fäßler, Peter Pimpl |
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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