Abstract
We describe production of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR) for 19F-NMR and single-molecule fluorescence (SMF) spectroscopy. We explain in detail steps shared between the two sample preparation strategies, including expression and isolation of A2AAR and assembly of A2AAR in lipid nanodiscs and procedures for incorporation of either 19F-NMR or fluorescence probes. Protocols for SMF experiments include sample setup, data acquisition, data processing, and error analysis. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022) and Sušac et al. (2018).