Marsango Sara, Varela María José, Milligan Graeme
Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, University Avenue, Glasgow, G12 8QQ, Scotland, UK.
Methods Mol Biol. 2015;1335:95-105. doi: 10.1007/978-1-4939-2914-6_7.
Fluorescence resonance energy transfer (FRET) is an approach widely used to detect protein-protein interactions in live cells. This approach is based on the sensitization of an "acceptor" molecule by the energy transfer from a "donor" when there is an overlap between the emission spectrum of the "donor" and the excitation spectrum of the "acceptor" and close proximity between the two fluorophore species (in the region of 8 nm). Various methods exist to quantify FRET signals: here, we describe the application of homogeneous time-resolved FRET (htrFRET) combined with Tag-lite™ technology and its application to determine not only protein-protein interactions but also the capability of GPCR mutant variants to form homomers compared to the wild type GPCR within the plasma membrane of transfected cells.
荧光共振能量转移（FRET）是一种广泛用于检测活细胞中蛋白质-蛋白质相互作用的方法。该方法基于当“供体”的发射光谱与“受体”的激发光谱有重叠且两种荧光团种类紧密接近（在8纳米范围内）时，通过从“供体”进行能量转移来敏化“受体”分子。存在多种量化FRET信号的方法：在此，我们描述了均相时间分辨FRET（htrFRET）与Tag-lite™技术相结合的应用，及其不仅用于确定蛋白质-蛋白质相互作用，还用于确定与野生型GPCR相比，GPCR突变变体在转染细胞的质膜内形成同聚体的能力。
