Study describes confocal and multi-photon fluorescence resonance energy transfer microscopy to characterise the dimerisation of transcription factors in living mouse cells
Details of a study describing the use of confocal and multi-photon Fret (fluorescence resonance energy transfer) microscopy techniques to characterise the dimerisation of transcription factors in the nucleus of living mouse pituitary cells have been published in a Bio-Rad application note.
Although several in vitro biochemical methods exist to study protein-protein interactions, it is not clear to what extent they accurately reflect behaviour in living cells.
Using a Bio-Rad Radiance2100 system, Periasamy of the University of Virginia, USA, describes the molecular behaviour of CFP- and YFP-tagged C/EBPa transcription factor and the use of Fret to quantify the formation of transcription factor C/EBPa dimers in the nucleus of these living cells.
Fret involves the transfer of energy from a donor fluorophore to an acceptor fluorophore resulting in emission from the acceptor as a result of donor excitation.
For transfer to take place the donor and acceptor molecules must be very close (<10nm) and measurement of the Fret signal provides spatial information about the two molecules at a resolution beyond the limits of conventional microscopy.
Dr Periasamy describes the advantages of using multi-photon Fret in work with living cells adopting a Coherent Ti:sapphire laser system.
The wide tuning range (700-1000nm) enables excitation of most fluorophore pairs while excitation in the infrared region causes significantly less intracellular damage.
The authors have also developed software dedicated to correcting contamination of the Fret signal by spectral bleed-through and fluorophore expression level variation in living cells and tissues.
The full details of the study may be read in Bio-Rad's Application Note 36, which is available free of charge.
The Radiance2100 confocal and multi-photon system is available exclusively from Bio-Rad Cell Science Division.
