Liposome preparation and binding assay

For liposome preparation, 10 mg 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids (Avanti Polar Lipids) were dissolved in a 1:1 methanol/chloroform mixture and lyophilized as a thin lipid film. The film was then rehydrated with 1 ml buffer-detergent mix, containing 1% Triton-X 100 in sample buffer. After dissolving, the mixture was incubated in a glass vial with 10% (w/v) Bio-Rad Bio-Beads SM-2 Adsorbent Media (Cat No. 152-3920) for 30 min at 4 degrees. Subsequently, Bio-Beads were added to a total of 30% (w/v), and the mixture was incubated overnight at 4 °C to remove all traces of detergent. As an alternative, we also prepared liposomes by the extrusion method, without using detergents: a suspension of 10 mg/ml POPC was homogenized by passing 21 times through a polycarbonate membrane with a 0.2 μm pore size in a mini extruder (Avanti Polar Lipids).

For the binding assay, the supernatant was diluted with sample buffer complemented with 5 mM CaCl₂ and 0.5 M KCl and incubated with α-LTX at final concentrations of 0.1 mg/ml liposomes and 0.02 mg/ml α-LTX for four hours at 310 K. The membrane incorporation of α-LTX was then analyzed by negative stain EM.