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However, as any other methodologies, it has its limitations and drawbacks and is
constantly being improved with regard to experimental design, instrumentation, and
reagents. For example, in some receptor-receptor interaction studies, because the ef-
ficiency of energy transfer is tightly dependent on proper orientation of the donor and
acceptor dipoles, conformational states of the fusion proteins may fix the dipoles into a
geometry that is unfavorable for energy transfer. Thus, two receptors may form het-
eroreceptor complexes without producing any significant BRET signal. Thus, a neg-
ative result with BRET does not necessarily mean absence of the heteroreceptor
complex. Furthermore, the luminescent and fluorescent protein fused to the candidate
protomers may affect their interaction and alter their subcellular localization, protein
folding, and receptor function. Moreover, as with any technique that involves transient
transfection, overexpression of the protomers may bring misleading results. Therefore,
such drawbacks limit its use today and lead to a demand for proper controls. It is now
easier to keep the receptor protomer expression levels within physiologically relevant
ranges, thanks to the improvement of donor/acceptor protein quantum yield (e.g., the
newmutated Rluc8). On the other hand, BRET
2
presents various advantages compared
to standard biochemical procedures to study receptor-receptor interactions that require
cell-invasive processes such as solubilization and coimmunoprecipitation, which in-
cludes even the previously developed FRET technique. BRET
2
, as opposed to FRET,
does not require the excitation of the donor with an external light source. Therefore, it
does not suffer from problems usually associated with autofluorescence, light scatter-
ing, photobleaching, and/or photoisomerization of the donor moiety, which results in
an overall improved signal-to-noise ratio when compared to earlier versions of the res-
onance energy transfer technologies. Also, the absence of contamination of the light
output by the incident light results in a very low background in BRET
2
assays, thereby
permitting the detection of small changes in the BRET
2
signal as compared to FRET.
Also, the BRET
2
signal is a ratiometric measurement, which can help to reduce data
variability caused by fluctuations in light output due to variations in assay volume, cell
types, number of cells per well, and/or signal decay across a plate. Finally, the coelen-
terazine derivative DeepBlueC or coelenterazine 400a used in BRET
2
is membrane
permeable and nontoxic, which makes BRET
2
an ideal assay technology for live cell
assays. DeepBlueC penetrates the cell membrane in seconds to activate Rluc8 emis-
sion. With respect to receptor-receptor interaction mechanisms and signaling research,
its capabilities have now significantly reached beyond studying GPCRs. The BRET
2
assay technology is used successfully for a wide range of assay types including GPCR-
beta-arrestin assay for monitoring GPCR activity, tyrosine kinase receptor activation,
Ca
2
þ
and cAMP detection, apoptosis assay, kinase activity, and protease activity.
Acknowledgments
This work has been supported by the Swedish Medical Research Council (04X-715), Telethon
TV3's La Marat´ Foundation 2008, and Hj¨rnfonden to KF; by grants from the Swedish Royal
Academy of Sciences (Stiftelsen B. von Beskows Fond and Stiftelsen Hierta-Retzius
