Mitochondrial Medicine pp 395-419
The Use of FLIM-FRET for the Detection of Mitochondria-Associated Protein Interactions
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 wordsFLIM FRET Protein–protein interactions Interactions at membrane Fluorescence lifetime imaging mCerulean3 Fluorescence proteins
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