Abstract
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.
Original language | English (US) |
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Pages (from-to) | 132-139 |
Number of pages | 8 |
Journal | Brain Research |
Volume | 1091 |
Issue number | 1 |
DOIs | |
State | Published - May 26 2006 |
Keywords
- Fluorescence resonance energy transfer
- Green fluorescent protein
- microscopy
- Protein-protein interaction
- Spectra FRET
- Spectroscopy
ASJC Scopus subject areas
- Neuroscience(all)
- Clinical Neurology
- Developmental Biology
- Molecular Biology