Extended Data Figure 3 |. Expression and purification of the MIPS521-ADO–A1R–G_i2 complex.

a, Expression and purification flowchart for the A1R–G_i2 complex. A1R and the G_i2 heterotrimer with Gβ₁γ₂ were expressed separately in insect cell membranes. Addition of ADO (1 mM) and MIP521 (100 nM) initiated complex formation, which was solubilised with 0.5% (w/v) lauryl maltose neopentyl glycol and 0.05% (w/v) cholesteryl hemisuccinate. Solubilised A1R and A1R –G_i2 complex was immobilised on Flag antibody resin. Flag-eluted fractions were purified by size-exclusion chromatography (SEC). b, Representative SDS–PAGE/western blot of the purified A1R–G_i2 complex. An anti-His antibody was used to detect Flag–A1R-His and Gβ₁-His (red) and an anti-G_i2 antibody was used to detect Gα_i2 (green). Experiment was performed three times with similar results c, Representative SDS–PAGE/Coomassie blue stain of the purified complex concentrated from the Superdex 200 Increase 10/30 column. Experiment was performed three times with similar results d, Representative elution profile of Flag-purified complex on Superdex 200 Increase 10/30 SEC. Experiment was performed three times with similar results