Preparation of unilamellar liposomes
Suzanne R Pfeffer, Ayan Adhikari
Abstract
We present here a protocol for preparing liposomes that can be used to monitor binding of proteins to vesicles of various membrane curvatures. We used this to study the association of the PPM1H phosphatase with highly curved membranes due to its N-terminal amphipathic helix.
Steps
Make lipid stocks
All lipids were purchased from Avanti Polar Lipids. PC and PS were obtained as 10mg/ml
chloroform solutions. PI, PI(4)P and cholesterol sulfate were obtained as powders. Dissolve in chloroform to prepare 2mg/ml, 1mg/ml and 25mg/ml stock solutions respectively.
Prepare the lipid mixture
Prepare unilamellar vesicles by mixing:
940µL of (18:0-20:4)PC
470µL of (18:0-20:4)PI
60µL of (18:0-18:2)PS
140µL of (18:1)PI(4)P
26µL of cholesterol sulfate
from the respective stock solutions in a glass vial. The lipid composition of the liposomes is in the ratio (mol %) 78:7:5:1:9 to represent the mammalian cell Golgi composition (Fasimoye et al., 2023).
Dry the above-mentioned lipid mixture in chloroform under nitrogen flow by pointing a Pasteur pipette into the glass vial. This vial is subsequently incubated under house vacuum for at least 1h 0m 0s.
Resuspend the dried lipids by pipetting up and down in 1ml of resuspension buffer (50mM HEPES pH 7.5, 120mM KCl)
Liposome preparation
Sonicate the liposome suspension by two brief 0h 0m 5s cycles of bath sonication followed by sequential extrusion through 0.4, 0.1 and 0.05 µm pore size polycarbonate filters for 21 times using a hand extruder.
Liposome storage
The final lipid concentration of the liposome suspension will be 15mM; store at 4°C for a maximum of 2 weeks. Small liposomes are best used close to the time of their preparation.
