Mitochondrial Medicine pp 395-419

Part of the Methods in Molecular Biology book series (MIMB, volume 1264) | Cite as

The Use of FLIM-FRET for the Detection of Mitochondria-Associated Protein Interactions

  • Elizabeth J. Osterlund
  • Qian Liu
  • David W. Andrews
Protocol

Abstract

Fluorescence lifetime imaging microscopy–Förster resonant energy transfer (FLIM-FRET) is a high-resolution technique for the detection of protein interactions in live cells. As the cost of this technology becomes more competitive and methods are devised to extract more information from the FLIM images, this technique will be increasingly useful for studying protein interactions in live cells. Here we demonstrate the use of the ISS-Alba FLIM/FCS confocal microscope, which was custom-built for supervised automation of FLIM data acquisition. We provide a detailed protocol for collecting and analyzing good FLIM-FRET data. As an example, we use FLIM-FRET to detect the interaction between BclXL and Bad at the mitochondrial outer membrane in live MCF7 breast cancer cells.

Key words

FLIM FRET Protein–protein interactions Interactions at membrane Fluorescence lifetime imaging mCerulean3 Fluorescence proteins 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Elizabeth J. Osterlund
    • 1
  • Qian Liu
    • 3
  • David W. Andrews
    • 1
    • 2
  1. 1.Department of BiochemistryUniversity of TorontoTorontoCanada
  2. 2.Odette Cancer Research ProgramSunnybrook Research InstituteTorontoCanada
  3. 3.Department of BiochemistrySunnybrook Research InstituteTorontoCanada

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