Aug 25, 2023

Liposome preparation

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr612ovmk/v1
  • 11. Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 22. Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
xinbo.wang
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr612ovmk/v1
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. Liposome preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr612ovmk/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 19, 2022
Last Modified: May 31, 2024
Protocol  Integer ID: 68882
Keywords: Liposome, LRRK2, Lipid nanotube, ASAPCRN, ps lipid nanotube, liposome, lrrk2 binding
Abstract
This protocol details methods for the preparation of 100% PS liposome and GC/PS lipid nanotubes used for LRRK2 binding, tubulation assays.
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Materials
Brain PSAvanti Polar Lipids, Inc.Catalog # 840032
Galactosylceramide (GC)Avanti Polar Lipids, Inc.Catalog #860546P
Rhod-PEAvanti Polar Lipids, Inc.Catalog #810150
Cy5-PEAvanti Polar Lipids, Inc.Catalog #810345

Solutions to prepare:

Liposome buffer:

AB
HEPES (7.4)20 mM
KCl100 mM
TCEP0.5 mM
Liposome preparation
1h 5m
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 01:00:00 .
1h
Rehydrate the dried lipid films in liposome buffer at a final concentration of 1 mg/mL (~1.2 millimolar (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 (500 x g for 00:05:00 ) and store in the dark at 4 °C to avoid photooxidation.
5m