Bret

Renilla luciferase is able to oxidize substrates such as h -coelenterazines or DeepBlueC, a reaction that generates bioluminescence with peaks in the emission spectra either at 475 nm (h-coelenterazine) or 395 nm (DeepBlueC) [47, 48]. In order to use bioluminescence as a donor for RET, acceptor fluorophores with suitable excitation and emission spectra need to be used. Typically, enhanced yellow fluorescent protein (eYFP) is used as an acceptor for h-coelenterazines (BRET1), whereas GFP2 is used as an acceptor for DeepBlueC (BRET2) -48] . BRET2 has the advantage that the emission of DeepBlueC does not overlap with the emission spectra of GFP2 . resulting in reduced bleed-through of the donor into the acceptor channel. Thus, for accurate measurement of the absolute BRET efficiency, BRET2 is preferable. However, a disadvantage of BRET2 is the lower quantum yield and therefore the requirement to use higher amounts of enzyme, either by increasing the sample size, or more typically, by increasing the expression [51]. For the latter case, overexpression artifacts have to be considered with BRET2, even more than when BRET1 is utilized. For luciferase assays such as BRET, the time point of substrate application prior to the actual measurement is very critical for the amount of bioluminescence measured, due to the depletion of substrate over time. This has been problematic with respect to repeated long-term measurements, and the difficulty has led to the development of coelenterazine as a precursor, which is taken up by cells and then later enzymatically releases h - coelenterazine.

BRET is most often measured by means of ratiometric detection of YFP or GFP2 emission over donor emission derived from the bioluminescence emitting cleavage of suitable substrates by the luciferase. The detection occurs via a sensitive luminometer, typically based on photomultiplier tubes. As the bioluminescence intensity is orders of magnitudes lower than that of fluorescence derived from direct excitation of fluorescence by high power excitation light sources, typically, luminescence is detected in multicellular preparations [49] .

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