The high fluorescence quantum yield of TagGFP2 along with the high molar extinction coefficient of TagRFP and excellent overlap of donor emission and acceptor excitation spectra result in highly effective FRET between these fluorescent proteins.
The calculated Forster distance (R0= 5.7 nm) for the TagGFP2-TagRFP pair is one of the largest among the values reported.
At the same time, since TagGFP2 and TagRFP emission peaks are spaced by as much as 78 nm, the emission signal for these two proteins can be easily separated in any imaging system.
Shifting the wavelengths toward the red part of the spectrum (comparing to traditional cyan and yellow FRET partners) reduces input of cellular autofluorescence.
The significantly increased quantum yield of TagRFP is highly beneficial for acceptor-based ratiometric FRET studies.
High pH-stability of the both proteins allows using this pair for imaging in acidic organelles.
The combined advantages suggest that the TagGFP2-TagRFP is one of the most efficient green/red FRET couple available to date for ratiometric FRET analyses.
As an additional advance, TagRFP and TagGFP2 proteins derive from different marine sources and therefore luck the ability to form heterodimers.
It ensures zero background for FRET analysis that may not be the case for weakly dimerizing FRET pairs consisting of highly homological fluorescent proteins.
We believe that advantageous characteristics make TagGFP2-TagRFP one of the preferable FRET pairs to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors of various specificity (see demonstration of FRET-based caspase-3 apoptosis sensor
The excitation wavelength required to visualize FRET changes of the TagGFP2-TagRFP pair by ratio-imaging is provided by an ordinary FITC/GFP excitation filter or ubiquitous 488 nm laser line, and the two emission signals are acquired using a 500-530 nm (FITC/GFP emission filter) bandpass filter and a 560-600 nm bandpass filter (Cy3/DsRed emission filter) or a 560LP longpass filter.