Lipid-in-Oil Mixture Preparation Protocol

The preparation of the lipid-in-oil mixture is based on published protocols^{27 (link),71 (link)}. We use POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids, Inc.) with 1% DSPE-PEG(2000) biotin (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000], Avanti Polar Lipids, Inc.; both 25 mg/ml in chloroform) and give 77 µl thereof in a 10 ml vial with 600 µl chloroform. In experiments with labeled membranes, 3 µl DOPE-ATTO655 (0.1 mg/ml in chloroform) is added. While being mixed on a vortex mixer, 10 ml of a silicon oil and mineral oil (Sigma Aldrich, M5904) mix (4:1 ratio) is slowly added to the lipid solution. Since the lipids are not fully soluble in this mix of silicon oil, mineral oil, and chloroform, the resulting liquid is cloudy.