Cholesteryl hemisuccinate

For protein purification, 2 mM ATP (Sangon Biotech) and 2 mM MgCl₂ were added to the thawed suspension, and the mixture was incubated with additional 1% (wt/vol) DDM (Bluepus) and 0.2% (wt/vol) cholesteryl hemisuccinate (Anatrace) at 8 °C for 2 h for membrane solubilization and protein extraction. After ultracentrifugation at 45,000 rpm for 45 min (Beckman Type 70 Ti), the supernatant was incubated with the anti-Flag M2 affinity gel (Sigma) on ice for at least 40 min. Then, the resin was washed by 30 mL of wash buffer containing 50 mM Tris⋅HCl (pH 7.5), 150 mM NaCl, and 10% (vol/vol) glycerol, with 0.02% GDN (wt/vol; Anatrace) for ATP8B1–CDC50A or 0.06% digitonin (wt/vol; Apollo Scientific) for ATP8B1–CDC50B. The protein was eluted with 6 mL of wash buffer plus 200 µg/mL Flag peptide. The samples were further concentrated and purified by SEC on a Superose 6 Increase 10/300 or Superdex 200 Increase 10/300 gel-filtration column (GE Healthcare), pre-equilibrated with SEC buffer (50 mM Tris⋅HCl, pH 7.5, 150 mM NaCl, and 0.02% GDN or 0.06% digitonin). The purified samples were analyzed by SDS-gel electrophoresis with a commercial precast acrylamide gel (with a gradient from 4 to 20%). The peak fractions were collected, concentrated to 4 to 6 mg/mL, frozen in liquid nitrogen, and stored at −80 °C before use.