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Investigating Protein–Protein Interactions in the Plant Endomembrane System Using Multiphoton-Induced FRET-FLIM

  • Jennifer SchobererEmail author
  • Stanley W. BotchwayEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)

Abstract

Real-time noninvasive fluorescence-based protein assays enable a direct access to study interactions in their natural environment and hence overcome the limitations of other methods that rely on invasive cell disruption techniques. The determination of Förster resonance energy transfer (FRET) by means of fluorescence lifetime imaging microscopy (FLIM) is currently the most advanced method to observe protein–protein interactions at nanometer resolution inside single living cells and in real-time. In the FRET-FLIM approach, the information gained using steady-state FRET between interacting proteins is considerably improved by monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the presence of the acceptor is evidence for a direct physical interaction. The combination of confocal laser scanning microscopy with the sensitive advanced technique of time-correlated single photon counting allows the mapping of the spatial distribution of fluorescence lifetimes inside living cells on a pixel-by-pixel basis that is the same as the fluorescence image. Moreover, the use of multiphoton excitation particularly for plant cells provides further advantages such as reduced phototoxicity and photobleaching. In this protocol, we briefly describe the instrumentation and experimental design to study protein interactions within the plant endomembrane system, with a focus on the imaging of plant cells expressing fluorescent proteins and acquisition and analysis of fluorescence lifetime resolved data.

Key words

Fluorescence imaging Microscopy Proteins Excited-state lifetime Multiphoton TCSPC FRET FLIM Golgi N-glycan processing enzymes 

Notes

Acknowledgements

This work was funded by the Austrian Science Fund (FWF): J2981-B20 (to J.S.), and Oxford Brookes University. Access to the Central Laser Facility, Rutherford Appleton Laboratory, was funded by a Science and Technology Facilities Council Program Access grant. We thank Chris Hawes for carefully reading the manuscript.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Applied Genetics and Cell BiologyUniversity of Natural Resources and Life Sciences, BOKU ViennaViennaAustria
  2. 2.Rutherford Appleton Laboratory, Central Laser FacilityResearch Complex at Harwell, Science and Technology Facilities CouncilHarwell-Oxford, DidcotUK

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