GFP-based FRET analysis in live cells.
Fluorescence resonance energy transfer (FRET) is a widely utilized optical technique for measuring small distances of 1-10 nm in live cells. In recent years, its application has been greatly popularized by the discovery of green fluorescent protein (GFP) and many improved variants which make good donor-acceptor fluorophore pairs. GFP-based proteins are structurally stable, relatively inert, and can be reliably attached to points of interest. The combination of easy access to the GFP-based FRET technique and its obvious usefulness in many applications can lead to complacency. Potential problems such as light contaminants, e.g., bleed-through and cross-talk, and inconsistent donor and acceptor concentrations are easily overlooked and can lead to errors in FRET calculation and data interpretation. In this article, we outline possible pitfalls of GFP-based FRET and approaches that address these issues, including a "Spectra FRET" technique that can be easily applied to live cell studies.
Takanishi
Christina L
CL
Department of Physiology and Membrane Biology, University of California School of Medicine, One Shields Avenue, Davis, CA 95616, USA.
Bykova
Ekaterina A
EA
Cheng
Wei
W
Zheng
Jie
J
D000818:Animals; D002477:Cells / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans
D049452:Green Fluorescent Proteins