Liposome preparation
Pietro De Camilli, Xinbo Wang
Abstract
This protocol details methods for the preparation of 100% PS liposome and GC/PS lipid nanotubes used for LRRK2 binding, tubulation assays.
Attachments
Steps
Liposome preparation
Dissolve lipid mixtures with chloroform in glass vials in moles percent as follows:
- PS liposomes: 99.5% brain PS:0.5% Rhod-PE.
- GC/PS nanotubes: 39.5% Galactosylceramide:60% brian PS:0.5% Cy5-PE.
Evaporate chloroform under a stream of nitrogen gas to produce a lipid film on the glass surface.
Dry the lipid film in a vacuum oven for 1h 0m 0s.
Rehydrate the dried lipid films in liposome buffer at a final concentration of 1mg/mL (~1.2millimolar (mM)).
For PS mixtures, form the liposomes by three freeze (liquid N2)–thaw (37°C water bath) cycles.
For GC/PS mixtures, form the lipid nanotubes by a brief vortexing instead of freeze-thaw cycles.
Remove large aggregates by a brief centrifugation (500x g,0h 0m 0s for 0h 5m 0s) and store in the dark at 4°C to avoid photooxidation.
