Detection of heteromerization of more than two proteins by sequential BRET-FRET

Combining BRET and FRET in the Sequential BRET-FRET (SRET) new technique permits heteromers formed by three different proteins to be identified. In SRET experiments, the oxidation of a Rluc substrate triggers acceptor excitation by BRET and subsequent energy transfer to a FRET acceptor. Thus, SRET requires the co-expression of three fusion proteins, one coupled to Rluc, another conjugated with GFP2 or YFP, and the third with YFP or DsRed. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will enable us to better understand how signals are integrated at the molecular level.