2.9. Liposome Binding Assay

Liposomes were prepared while using the standard method by dissolving DPPS in the chloroform and methanol (3:1) mixture. Subsequently, the organic solvent was removed by rotary evaporation, followed by vacuum pump for about half an hour. The dried lipid film was re-suspended in 20 mM Tris-HCl buffer, pH 7.0, containing 100 mM KCl, 0.5 mM DTT, and 0.2 mM EDTA, and then sonicated to produce small unilaminar vesicles with diameters that ranged from 15 to 50 nm. The quality of the final products was checked by negative-staining electron microscopy. The freshly prepared DPPS liposome was used for the PARN binding assay with a protin:DPPS molar ratio that ranged from 1:50 to 1:200. The final concentration of liposome was 200 μM, while that of PARN ranged from 1 μM to 4 μM. After 1.5 h incubation at room temperature, the mixture was centrifuged at 15,000× g for 15 min. at 4 °C. The supernatant and pellet fractions were both analyzed by SDS-PAGE electrophoresis and western blot.