Tla120 fixed angle rotor

Lipid mixtures were dried under nitrogen gas and then under vacuum for 1 h at room temperature. Dried lipids were hydrated liposome-binding buffer (20 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 1 mM DTT) for 60 min at 37°C, vortexed for 1 min, and subjected to three rounds of freeze-thaw cycles with liquid nitrogen. To remove protein aggregates, the protein solution was subjected to centrifugation using a TLA120 fixed angle rotor (Beckman) at 55,000 rpm for 15 min at 4°C before use. 10 µg of proteins were incubated with 50 nmol liposomes for 10 min at room temperature, and the mixture was centrifuged at 55,000 rpm for 30 min at 20°C using a TLA120 fixed angle rotor (Beckman). The resultant supernatant and pellet were subjected to SDS–PAGE, and the proteins and lipids were stained with Coomassie blue. The intensities of individual bands were quantified with Fiji.