Abstract
Bioluminescence resonance energy transfer (BRET) is a prominent biophysical technology for monitoring molecular interactions, and has been widely used to study protein-protein interactions (PPI) in live cells. This technology requires proteins of interest to be associated with an energy donor (i.e., luciferase) and an acceptor (e.g., fluorescent protein) molecule. Upon interaction of the proteins of interest, the donor and acceptor will be brought into close proximity and energy transfer of chemical reaction-induced luminescence to its corresponding acceptor will result in an increased emission at an acceptor-defined wavelength, generating the BRET signal. We leverage the advantages of the superior optical properties of the NanoLuc(®) luciferase (NLuc) as a BRET donor coupled with Venus, a yellow fluorescent protein, as acceptor. We term this NLuc-based BRET platform "BRET(n)". BRET(n) has been demonstrated to have significantly improved assay performance, compared to previous BRET technologies, in terms of sensitivity and scalability. This chapter describes a step-by-step practical protocol for developing a BRET(n) assay in a multi-well plate format to detect PPIs in live mammalian cells.
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