Comparison of BRET and FRET

Fluorescence resonance energy transfer (FRET), in which both the donor and acceptor molecules are fluorescent proteins or chemical probes, is a very popular biochemical technique employed in a variety of applications [8]. Several FRET pairs have been well studied and allow for a range of selection in terms of fluorescence excitation-emission wavelengths. In FRET studies, there is no need for addition of substrate to generate a signal. High signal levels can be achieved by selecting suitable fluorescent probes. In addition, because of the availability of extensive literature in this field, FRET has become a method of choice for many applications such as drug screening [9], in vivo biochemical analysis [10], receptor activation studies [11], metabolic screening [12], protein folding, [13] etc. In recent times, BRET has slowly gained in popularity over FRET. The former has several advantages as well as some limitations when compared to FRET. The main advantage ofBRET over FRET is that it does not require an excitation source and therefore is a better option for analysis of cells that are damaged by excitation light or are photoresponsive. The problem of photobleaching of fluorophores, as in the case of FRET, is avoided when employing BRET. In addition, the contribution of cell autofluorescence to the background becomes insignificant when the cells are assayed using BRET. While spectral separation between donor and acceptor excitation is needed in FRET to avoid problems with the possibility of exciting both the fluorophores, this problem is eliminated in BRET because the light emission from the donor occurs as a result of a chemical reaction. It should be noted that BRET biosensing systems can be designed by employing molecular biology tools, such as gene fusion, to create protein chimeras where both the donor and the acceptor molecule are part of the same protein molecules [6]. In these cases, a fusion protein is constructed using molecular biology tools such that the bioluminescent donor is fused to one of the termini of the protein under study. At the other terminus the fluorescent protein is genetically fused. This also allows measurement of expression levels of donor and acceptor fusion partners in a BRET pair independently, in contrast to FRET, where the acceptor can get excited to some extent with donor excitation. This is especially true in cases where green fluorescent protein (GFP) mutants are employed as FRET pairs. The knowledge of relative levels of expression of the fusion partner allows for a proper comparison between all the experiments performed, which in turn allows for the examination of other factors that may affect the background and thus the protein expression levels.

A major disadvantage of BRET over FRET is that the BRET signal can be weak. This can be overcome by using highly sensitive instruments in conjunction with the integration over a long period of time of the BRET signal generated as a result of the assay. There is no question that BRET is a powerful technique; however, to date only a few BRET donors have been identified and characterized. Unfortunately, this limits the choice of acceptors and the wide applicability of the method.

6.4 Examples of BRET Donor-Acceptor Pairs | 99

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  • jon appling
    How bret differ from fret?
    21 days ago

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