Sep 07, 2023
Preparation of unilamellar liposomes
- Ayan Adhikari1,2,
- Suzanne Pfeffer1,2
- 1Stanford University School of Medicine;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
External link: https://doi.org/10.1073/pnas.2315171120
Protocol Citation: Ayan Adhikari, Suzanne Pfeffer 2023. Preparation of unilamellar liposomes. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3ke7v4o/v1
Manuscript citation:
Wondwossen M. Yeshaw, Ayan Adhikari, Claire Y. Chiang, Herschel S. Dhekne, Paulina S. Wawro, Suzanne R. Pfeffer (2023) Localization of PPM1H phosphatase tunes Parkinson’s disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis.Proceedings of the National Academy of Sciences of the United States of America doi: 10.1073/pnas.2315171120
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: September 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 87516
Keywords: ASAPCRN, unilamellar liposome, preparation of unilamellar liposome, preparing liposome, vesicles of various membrane curvature, curved membrane, various membrane curvature, ppm1h phosphatase, association of the ppm1h phosphatase, terminal amphipathic helix, amphipathic helix, vesicle, binding of protein, protein
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000463
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.
Materials
1.(18:0-20:4)PC (1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine)
[Avanti #850469]
2.(18:0-20:4)PI (1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol)
[Avanti #850144]
3.(18:0-18:2)PS (1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine)
[Avanti #840063]
4.(18:1)PI(4)P {1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4'-phosphate)}
[Avanti #850151]
5. Cholesterol sulfate [Avanti #700016]
6. 0.4 µm pore size polycarbonate filters [Avanti #610007]
7. 0.1 µm pore size polycarbonate filters [Avanti #610005]
8. 0.05 µm pore size polycarbonate filters [Avanti #610003]
9.Chloroform [Fischer Chemical #C298-500]
10.Hand held mini extruder with heating block [Avanti # 610000)
Troubleshooting
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:
940 µL of (18:0-20:4)PC
470 µL of (18:0-20:4)PI
60 µL of (18:0-18:2)PS
140 µL of (18:1)PI(4)P
26 µL 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 01:00:00 .
1h
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 00:00:05 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.
5s
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.
Protocol references
1. R. Fasimoye et al., Golgi-IP, a tool for multimodal analysis of Golgi molecular content. Proc Natl Acad Sci U S A 120, e2219953120 (2023)