Type-3 BRET, an improved competition-based bioluminescence resonance energy transfer assay
Felce JH, Knox RG, Davis SJ. (2014), Biophys J. 106, L41-3
We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membrane protein, CD86, and two G protein-coupled receptors β2AR and mCannR2, all of which behave as dimers in these conventional assays, exhibit monomeric behavior in an improved competition-based type-3 BRET assay designed to circumvent such artifacts.
Key figure: Application of type-3 BRET to type-I TM proteins and GPCRs
Reduced BRETeff at a given expression level was observed for the dimers CD28, CD80, and GABAbR2 in the presence of competitor (A and C), but not for the monomers CD2 and CD86 (B). The GPCRs mCannR2 and β2AR did not exhibit a change in BRETeff/expression relationship upon addition of competitor (C). Data are combined from three independent experiments in all cases except CD86, which constitutes four experiments.