BRET assay

Like FRET, today BRET is predominantly used in biological sciences, especially in the monitoring of protein-protein interactions such as hormone-receptor interaction [223, 224] and protein-DNA interaction in living systems. However, BL resonance energy transfer can also be applied in immunoassays by using for instance a peptide-tagged luciferase and a fluorescein-labeled antipeptide antibody [225]. The development of more BRET assays for small-molecule analytes is thus awaited. 

Figure 14.9 (a), (b) Discrimination between negative and positive control of the BRET assay technology. 

After 18 h, cells are washed twice with PBS, detached with Versene, and resuspended in BRET assay buffer. 

The absence of an external light source in BRET assays is also advantageous in the development of a more portable instrument. 

BRET assay in a microfluidic device for quantifying thrombin-catalyzed peptide. 

There is a wealth of biochemical data on the activation of G proteins by receptors. Based on this very large body of evidence coming largely from experiments with cell membranes and reconstituted systems with the purified proteins, the now classical G protein cycle has been proposed (Bourne et at., 1990 Gilman, 1987). The core characteristic of this cycle is that the activated receptor causes GTP binding and subsequent dissociation of the heterotrimeric G protein into the active a. and fly subunits. FRET- and BRET-based assays have recendy been developed in order to investigate G protein activation in intact systems and also to assess whether G protein dissociation is indeed a requirement for G protein activation in the intact cell membrane. 

In conclusion, BRET offers the ability to directly study complex protein-protein interactions in living cells. There is no need for an excitation light source. Therefore, photosensitive tissue can be used for BRET, and problems associated with FRET-based assays such as photo-bleaching, autofluorescence, and direct excitation of the acceptor are eliminated. 

Since Rluc and GEP recombinantly fused proteins can be expressed in living cells, BRET is an interesting tool for monitoring molecular interactions in cell-based assays. BRET has been particularly used for the study of GPCRs by probing receptor oligomerization or activation. ... 

In this chapter, we describe protocols to set up BRET-based arrestin recruitment test systems. We provide suggestions for troubleshooting and identify experimental parameters that can be varied. We also discuss potential pitfalls and, importandy, some limitations of BRET as a quantitatively comparative method. Indeed, BRET is now widely used as a proximity-based assay to detect arrestin recruitment, but it is often overlooked that, besides proximity, the relative orientations of the probes crucially determines BRET efficiency. This somewhat complicates the accurate interpretation of BRET data, but careful setup and combination of different BRET systems hold promise for the study of yet underexplored questions in chemokine receptor signaling. 

The standard arrestin recruitment BILET protocol is similar for aU assay configurations, variations, and setup experiments. We thus give the standard protocol in this section and indicate the parameters that can or must be varied during setup or for specific questions. The standard protocol is also similar between BRET and BBJ3T, which use different energy donor and acceptor variants—minor variations between BBJ5T and BILET protocols are directly indicated in the protocol below, while specifics of BRET are dealt with in a separate paragraph below (Section 2.5.2). 

As outhned above, the efficacy of the BRET responses (BREToj x) cannot be equated with the efficacy of arrestin recruitment. If quantitative measures of arrestin recruitment efficacy are sought, this may be better served with the use of reporter complementation assays. These have successfully been used with chemokine receptors (Ikeda, Kumagai, Skach, Sato, Yanagisawa, 2013 Rajagopal et al., 2013). Note that these assays, while being quantitative, do not permit arrestin dissociation experiments, and that conformational information is lost. 

Alternative P-arrestin assays are BRET/FRET-based and have been suc-cessfiilly employed at both chemokine and nonchemokine receptors (Gilliland, Salanga, Kawamura, Trejo, Handel, 2013 Zhao et al., 2013). 

Bioluminescence resonance energy transfer (BRET) is a form of Eorster resonance energy transfer (RET). It is the non-radiative transfer of energy from an excited state donor to a ground state acceptor. The efficiency of energy transfer is highly dependent on the distance between the donor and acceptor moieties and their relative orientation with respect to each other. The photon emissions from the donor and receptor molecules during BRET reaction can be measured to quantify assay products. 

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