Comparison of BRET and FRET

Both methods, BRET and FRET, have been successfully applied to the field of GPCR research. The methods have unique advantages and disadvantages, which will be discussed only briefly in this context. Due to strong signal strength, FRET clearly has the advantage in terms of imaging where things happen. For certain fluorophores, FRET can be detected even between single molecules [53] ; though this has not yet been applied to GFP-based receptor studies, in the future it will likely be an important tool. FRET has demonstrated to be useful when analyzing receptor conformational changes as well as interaction characteristics with associating proteins. FRET can be measured by means of at least three reliable and quantitative techniques: Donor dequenching by acceptor photo bleaching (a method preferable to measure FRET efficiency), ratiometric detection of sensitized emission (preferable for resolving fast kinetics) and by fluorescence lifetime imaging (FLIM; a method ideally suited to image steady state FRET signals with subcellular resolution) [54]. As all three methods detect FRET by different means, the combination of all three methods can deliver accurate results regarding the occurrence of FRET, including its subcellular localization. In its current form, BRET relies only on ratiometric detection of light derived from acceptor and donor, and these measurements can be done with high sampling frequency. However, given that BRET measurements have to be done with typically at least 1000 cells per well, the kinetics of BRET responses are often limited by mixing time and always represent the average of a cell population. For very weak RET, BRET2 is probably more sensitive compared to FRET, due to the separated emission spectra from donor and acceptor. BRET assays are quite straightforward to perform, rely on automated luminescence readers, and offer the advantage of being able to measure many different experimental conditions within a short period of time [48]. It should come as no surprise that BRET has become a popular and powerful method by which receptor-receptor interactions can be studied, with a particular strength on generation of unbiased results (due to averaging and automation).

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