fluorescence resonance energy transfer - stanford university
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Fluorescence Resonance Energy Transfer (FRET)
Förster Radius The distance at which energy transfer is 50% efficient (i.e. 50% of excited donors are deactivated by FRET) is defined by the Förster Radius (R0).
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Applications of FRET in Biology
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Survey of FRET-Based Assays
• Protease activity• Calcium Ion measurements• cAMP• Protein tyrosine kinase activity• Phospholipase C activity• Protein kinase C activity• Membrane potential
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FRET probes conformational changes
Different conformation givesDifferent FRET signature
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FRET increasesIn both cases
Inter and IntramolecularForms of FRET withProteins
CFP-YFP good combo
Protein-Protein InteractionsIn cytoplasm andmembranes
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No FRET forNo overlap of donor emission,acceptor absorption
When FRET Occurs
No FRET forOrthogonal dipoleorientation
No FRET for moleculesmore than 10 nm apart
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Number of FRET Publications since 1989
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Fluorescence Resonance Energy Transfer -Detection of Probe Proximity
R0 typically 40-50 Angstroms50% transfer
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Typical Values of Ro
Donor Acceptor Ro (Å) Fluorescein Tetramethylrhodamine 55 IAEDANS Fluorescein 46 EDANS DABCYL 33 Fluorescein Fluorescein 44 BODIPY FL BODIPY FL 57 Fluorescein QSY 7 dye 61 Cy3 Cy5 53 CFP YFP 50
green red
GFPs and other colored “FPs have transformed FRET microscopy
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FRET Considerations:
1. Spectral overlap
2. Chromophore orientations
3. Distance dependence (Eff. ∼ 1/R6)
4. How to quantify?
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Practical Challenges to FRET Quantitation
• Emission from A contaminates D channel (filters)• Emission from D contaminates A channel• Unknown labeling levels for D and A• Signal variation due to bleaching
– Complicates kinetic studies– Bleaching rate of D can actually be slowed by FRET
Solutions:• Separately labeled D and A controls to define
bleedthrough• Acceptor destruction by photobleaching to establish • Dual wavelength ratio imaging to normalize away
variations in label levels and bleaching effects