What is CFP and YFP?
Abstract. The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking and tracing of specific proteins or cells or to determine protein interactions.
What is YFP CFP ratio?
Any signal in the FRET channel (CFPex/ YFPem) is therefore due to crosstalk of the CFP signal into this channel. This crosstalk (measured as a ratio of the CFP/YFP signal to the CFP/CFP signal) will be subtracted from the FRET signal measured in experimental cells. In our system it is usually 50-70%.
How do you choose a donor and acceptor in FRET?
Thus, in order to maximize the FRET signal, the researcher must choose the highest quantum yield donor, the highest absorbing acceptor, and fluorophores having significant overlap in their spectral profiles.
What makes a good FRET pair?
To maximize FRET E, a red-shifted FRET pair with improved optical properties including QY, EC and spectral overlap should be used. For a given FRET pair, the FRET E is proportional to the inverse sixth power of the distance between two fluorophores and works only over a distance shorter than 10 nm (Figure 1B).
What is the best FRET pair?
CFP-YFP is still one of the best and most used pairs to measure FRET. Other issues that can affect FRET measurements include: the brightness of a fluorophore pair, donor:acceptor stoichiometry, and cross-talk between the two fluorophore colours.
What is acceptor photobleaching?
Fluorescence resonance energy transfer (FRET) is an extremely effective tool to detect molecular interaction at suboptical resolutions. One of the techniques for measuring FRET is acceptor photobleaching: the increase in donor fluorescence after complete acceptor photobleaching is a measure of the FRET efficiency.
How do you Analyse a FRET?
One approach to detect FRET is to measure the fluorescence intensity ratio of the acceptor and the donor fluorophores observing the increase of the fluorescence intensity in the acceptor channel with the simultaneous decrease of the intensity in the donor channel.
How does the FRET technique work?
How Does FRET Work? The principle of FRET relies on the transfer of excitation energy of a donor fluorophore to a nearby acceptor fluorophore in a non-radiative fashion through long-range dipole-dipole interactions, when the distance separating them is 8 to 10 nanometers or less.
What is CFP–YFP fret and why is it important?
The analysis of CFP–YFP FRET on a cell-by-cell basis allows for a sensitive and highly rigorous assessment of protein interaction, and the large number of cells that can be examined by flow cytometry provides for a high degree of statistical confidence.
How to distinguish between YFP and CFP after excitation?
By using separate lasers to excite CFP and YFP, the detection of FRET was separated from that of YFP. Therefore, after direct excitation, the YFP emission spectrum remained constant in all transfected cells, whereas the emission spectrum of CFP varied with the extent of FRET in individual cells.
What is the relationship between fret and CFP fluorescence?
Thus, the fluorescence of CFP is quenched in proportion to the efficiency of FRET [41]. Therefore, FRET can be observed as a change in the amount of CFP fluorescence that results from the introduction or removal of its YFP counterpart.
How to assess donor CFP quenching during CFP–>YFP fret?
We have established a novel flow cytometric approach to assess donor CFP quenching during CFP–>YFP FRET, which can be used for the calculation of FRET efficiency and relative biological molecular distance between CFP and YFP moieties. This method can be used not only to analyze cells that expres …