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Monoclonal Antibody Fluorochrome FRET


FRET Pairs - Pacific Blue and FITC
- a CFP-YFP mimic

This type of FRET with conjugated monoclonal antibodies (mcab) allows the detection of receptors, protein interactions, conformational changes and receptors moving closer to each other classically with CD4 moving closer to CD3/TCR complex during T cell activation.

The labelling of murine thymocyte subpopulation bearing the CD8 receptor alpha-alpha homodimers make up 10% of the thymocyte population, although this population of cells also bear a high degree of CD8 made up of alpha-beta hetrodimers. These alpha homodimer receptors can be detected by tetramers or alpha-alpha specific antibodies which are not commercially available. The use of the same antibody clone to CD8 alpha chains conjugated to two fluorochromes that spectrally overlap e.g. Pacific Blue and FITC allows the detection and isolation of the CD8 alpha-alpha receptor by FRET sorting.

This type of FRET sorting of live cells can be done on a instrument with an violet diode and argon lasers, these two lasers excite Pacific Blue and FITC separately and also mimic the common FRET pairs CFP & YFP. Firstly the argon 488 nm laser excites the FITC which emits light in the argon laser 530 nm PMT. Next, violet diode excitation (405nm) of the Donor, Pacific Blue transfers energy non-radiatively to the Acceptor, FITC which then emits FRET derived energy at 530 nm in the violet diode 530nm PMT, see figure for pictorial explanation in Theory of FRET detection. The median fluorescence's of non-FRET control and FRET sample at channel 7.7 and 10.01, gives a FRET Efficiency Ratio of 0.3 according to the FRET Efficiency Ratio Formula.






Theory of FRET detection with
Pacific Blue and FITC

Pacific Blue-FITC FRET

FRET Efficiency Ratio Formula


by Gary Warnes. © Queen Mary, University of London 2007
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