Purification of N-terminally Tagged M2 Muscarinic Receptor

Human M₂ muscarinic receptor bearing the c-Myc or FLAG epitope at the N-terminus was expressed in Sf9 cells as described previously (Redka et al., 2014 (link); Shivnaraine et al., 2012 (link)) and in Appendix 1. The cells were harvested and solubilized in digitonin–cholate (0.86% digitonin, Wako Chemicals USA; 0.17% cholate, Sigma-Aldrich), and aliquots of the extract were removed for electrophoresis and binding assays. The receptor was purified in predominantly monomeric form by successive passage on DEAE-Sepharose, ABT-Sepharose, and hydroxyapatite. The final concentrations of digitonin and cholate were 0.1% and 0.02%, respectively. Purified receptor was stored at −75°C. Further details regarding the purification and the nature of the purified receptor have been described previously (Redka et al., 2014 (link)).