Imaging protein-protein interactions by Förster resonance energy transfer (FRET) microscopy in live cells.
This unit describes an acceptor-sensitized emission FRET method using a confocal microscope for image acquisition. In contrast to acceptor photobleaching, which is an end-point assay that destroys the acceptor fluorophore, the sensitized emission method is amenable for FRET measurements in live cells and can be used to measure changes in FRET efficiency over time. The purpose of this unit is to provide a basic starting point for understanding and performing the sensitized emission method with a simple teaching tool for live-cell imaging. References that discuss the vagaries of acquiring and analyzing FRET between individually tagged molecules are provided.
NIH/NIAID-LIG Imaging Facility, Rockville, Maryland.
Joseph A
JA
Brzostowski
NIH/NIAID-LIG Imaging Facility, Rockville, Maryland.
Tobias
T
Meckel
NIH/NIAID-LIG Imaging Facility, Rockville, Maryland.
Jiang
J
Hong
NIH/NIAID-LIG Imaging Facility, Rockville, Maryland.
Alice
A
Chen
NIH/NIAID-LIG Imaging Facility, Rockville, Maryland.
Tian
T
Jin
D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D038761:Photobleaching; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D008164:Luminescent Proteins; D011506:Proteins