TY - JOUR T1 - ThermoFRET: A novel nanoscale G protein coupled receptor thermostability assay functional in crude solubilised membrane preparations JF - bioRxiv DO - 10.1101/2020.07.07.191957 SP - 2020.07.07.191957 AU - David N Tippett AU - Brad Hoare AU - Tamara Miljus AU - David A Sykes AU - Dmitry B. Veprintsev Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/07/08/2020.07.07.191957.abstract N2 - Sensitive protein stability assays for membrane proteins are crucial for developing purification protocols, for structural and biophysical characterisation and drug discovery. Here, we describe a novel high-throughput 384-well FRET-based thermostability methodology, ThermoFRET, allowing for the ultrasensitive determination of G protein coupled receptor (GPCR) stability. This method measures FRET between a terbium-cryptate labelled GPCR and BODIPY-FL-Cystine, a thiolreactive dye that reacts with cysteine residues exposed upon protein unfolding in response to thermal denaturation. ThermoFRET is functional in crude solubilised membrane preparations, without protein purification and can detect receptor stabilising ligands, making it ideally suited for orphan receptor screening.Competing Interest StatementThe authors have declared no competing interest. ER -