For all experiments except membrane patch experiments, DMPC (dimyristoylphosphatidylcholine) and DMPG (dimyristoylphosphatidylglycerol) were purchased from Larodan Fine Chemicals AB (Malmö, Sweden). Lipid stocks were prepared by dissolving dry lipids in chloroform or chloroform:methanol (75:25) to a final concentration of 10 mg/ml. The solvent was evaporated under a stream of nitrogen gas before freeze-drying overnight at -52 °C under vacuum. The dried lipid mixture was stored air-tight at -20 °C until use.
For preparation of either liposomes or bicelles, the dried lipids were hydrated with either water or HBS (20 mM HEPES, 150 mM NaCl, pH 7.5) at a final concentration of 5-10 mg/ml, inverting at room temperature overnight to ensure that no unsuspended lipids remained in the glass vial. The formed multilamellar vesicles (MLVs) were subjected to freeze-thawing using liquid N2 and a warm water bath with 30 s of strong vortexing. The cycle was repeated 7 times. Small unilamellar vesicles (SUVs) were prepared by sonicating fresh MLVs using a probe tip sonicator (Branson Model 450, Sonics & Materials Inc. Vibra-cell VC-130) until the lipid suspension was clear, while avoiding overheating. Bicelles were made by adding n-dodecylphosphocholine (DPC) to the MLVs to a q-ratio of 2.84, while pipetting up and down until the solution became clear. The SUVs and bicelles were left at room temperature for at least a couple of hours before use.
For membrane patch experiments, DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPS (1,2dioleoyl-sn-glycero-3-phospho-L-serine) were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Lipid stocks containing DOPC and DOPS were prepared by dissolving dry lipids in methanol (hypergrade for LC-MS, Merck) to a final concentration of 10 mM with a DOPC:DOPS ratio of 9:1. When the lipids were completely dissolved, 0.05% DiD-C18 probe (Thermo-Invitrogen) was added. The lipid mixture was immediately used to make the lipid films for the patch experiments.
For preparation of either liposomes or bicelles, the dried lipids were hydrated with either water or HBS (20 mM HEPES, 150 mM NaCl, pH 7.5) at a final concentration of 5-10 mg/ml, inverting at room temperature overnight to ensure that no unsuspended lipids remained in the glass vial. The formed multilamellar vesicles (MLVs) were subjected to freeze-thawing using liquid N2 and a warm water bath with 30 s of strong vortexing. The cycle was repeated 7 times. Small unilamellar vesicles (SUVs) were prepared by sonicating fresh MLVs using a probe tip sonicator (Branson Model 450, Sonics & Materials Inc. Vibra-cell VC-130) until the lipid suspension was clear, while avoiding overheating. Bicelles were made by adding n-dodecylphosphocholine (DPC) to the MLVs to a q-ratio of 2.84, while pipetting up and down until the solution became clear. The SUVs and bicelles were left at room temperature for at least a couple of hours before use.
For membrane patch experiments, DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPS (1,2dioleoyl-sn-glycero-3-phospho-L-serine) were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Lipid stocks containing DOPC and DOPS were prepared by dissolving dry lipids in methanol (hypergrade for LC-MS, Merck) to a final concentration of 10 mM with a DOPC:DOPS ratio of 9:1. When the lipids were completely dissolved, 0.05% DiD-C18 probe (Thermo-Invitrogen) was added. The lipid mixture was immediately used to make the lipid films for the patch experiments.
