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Kinetic FRET


Alexa Fluor488-555 FRET

Antibodies conjugated with Alexa Fluor dyes such as Alexa Fluor 488 and Alexa Fluor 555 can also act as donor and acceptor fluorophores to detect FRET. Such Alexa Fluor labelling of 3-phosphoinositide dependent protein kinase 1 (PDK1) and phospholipase Cg1(PLCg1) on fixed cells allows FRET to measured in the 575nm argon laser channel (PE) dynamically over time enabling easy and meaningful display of the FRET signal due to PDK1 interaction with PLCg1. EGF stimulation of transiently transfected breast epithelial MDA-MB-231 cells can show that protein kinases such as PDK1 regulate phospholipase C, PLCg1 which has been implicated in cancer cell invasion.

Small differences often observed in FRET experiments show only small shifts when FRET and control are overlaid in flow cytometry histograms but can be enhanced by plotting data on a linear not a logarithmic scale, see figure.

This problem of displaying raw FRET data can be overcome by kinetic analysis of the FRET data or by displaying the FRET signal against Time. This can be done on a instrument with only an argon laser, with the Alexa Fluor-488 being excited by the laser and the Alexa Fluor555 conjugate is not excited by an argon laser but emits any FRET signal in the 575nm or PE channel on a standard flow cytometer, if the Alexa Fluor 555 is excited by Alexa Fluor-488 emission, see figure.

For a pictorial explanation of the Theory of FRET detection with a single laser, see figure.


C Raimondi, A Chikh, AP Wheeler, T Maffucci, M Falasca. A novel regulatory mechanism links PLCg1 to PDK1. J Cell Sci Epub May18 2012.





Theory of FRET detection with
Alexa Fluor488 & 555

Kinetic FRET

Histogram analysis

FRET Efficiency Ratio Formula


by Gary Warnes. © Queen Mary, University of London 2007
Institute of Cell and Molecular Science, The Blizard Building, Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, UK Tel: +44 (0)20 7882 2483, Fax: +44 (0)20 7882 2200