Delipidation and Purification of ApoA-I

DGUC-HDLs were delipidated as previously described (9 (link)). Briefly, 6 ml of ice-cold methanol:chloroform (1:1) was added to 1 ml of lyophilized DGUC-HDL, mixed, and incubated on ice for 30 min. Chilled methanol (4 ml) was added to the mixture, centrifuged (3000 rpm) for 10 min, removed supernatants, and repeated until pellet turned white. Chilled methanol (10 ml) was re-added, mixed, and recentrifuged. The pellet was dried under nitrogen, resolubilized with 6M guanidine HCl, and dialyzed in ammonium bicarbonate (10 mM) overnight. ApoA-I was isolated from delipidated HDL with 6M Urea by anion exchange chromatography with Q-Sepharose Fast Flow column coupled with a Q-Sepharose High-Performance column on an AKTA pure FPLC system (Cytiva). ApoA-I fractions were confirmed using Coomassie-based Aquastain (BulldogBio) on an SDS-PAGE gel and were dialyzed in ammonium bicarbonate (10 mM).