Jun 15, 2026

Generation of liposomes and proteoliposomes

DOI
https://dx.doi.org/10.17504/protocols.io.x54v95y6ml3e/v1
  • Elisabeth Holzer1
  • 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Elisabeth Holzer
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v95y6ml3e/v1
Protocol Citation: Elisabeth Holzer 2026. Generation of liposomes and proteoliposomes. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v95y6ml3e/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
-
Created: August 01, 2025
Last Modified: June 15, 2026
Protocol  Integer ID: 223994
Keywords: ASAPCRN, generation of liposome, liposome, proteoliposome, proteoliposomes this protocol
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol describes the generation of liposomes and proteoliposomes.
Materials
SEC300 buffer:

AB
HEPES pH 7.525 mM
NaCl300 mM
TCEP1 mM
SEC150 buffer:

AB
HEPES pH 7.525 mM
NaCl150 mM
TCEP1 mM
Lipids Used:

AB
LipidSupplier/Details
DOPCAvanti, Cat# 850375C
DOPEAvanti, Cat# 850725C
DOPSAvanti, Cat# 840035C
PI3PSigma-Aldrich, Cat# 850150P-500UG
Liver PISigma-Aldrich, Cat# 840042C-25MG
NBD-PESigma-Aldrich, Cat# 810155P-1MG
Rh-PEInvitrogen, Cat# L-1392
DGS-NTAAvanti, Cat# 790404C
SphingomyelinAvanti, Cat# 860584P-5MG
ATTO390-PEATTO-TEC, Cat# AD 390-161
DOPCAvanti Polar Lipids, Inc.Catalog #850375C
DOPE Avanti Polar Lipids, Inc.Catalog #850725C
DOPS Avanti Polar Lipids, Inc.Catalog #840035C
Liver PIMerck MilliporeSigma (Sigma-Aldrich)Catalog #840042C
NBD PEMerck MilliporeSigma (Sigma-Aldrich)Catalog #810155P
DGS-NTA Avanti Polar Lipids, Inc.Catalog #790404C
Sphingomyelin Avanti Polar Lipids, Inc.Catalog #860584P
ATTO390-PEATTO-TECCatalog #AD 390-161
CHAPSGlycon Biochemicals GmbhCatalog #D99009
Bio-Beads™ SM-2 ResinBio-Rad LaboratoriesCatalog #1523920
Equipment
19mm Nuclepore™ Polycarbonate Track-Etched Membranes
NAME
Whatman
BRAND
10419504
SKU
https://scientificfilters.com/whatman/membranes-track-etched-polycarbonate-800310
LINK

Equipment
Mini Extruder (Avanti, Cat# .
NAME
Avanti
BRAND
610024
SKU
https://avantiresearch.com/product/610024
LINK



Preparation of Lipid Films
1h
Mix desired lipid composition (as specified in figure legends) in a glass vial using lipids dissolved in chloroform.
Dry the mixture under a stream of argon.
Further desiccate under vacuum for 01:00:00 to form a dry lipid film.
1h
Lipid Film Rehydration
2m
Rehydrate lipid film with appropriate buffer depending on application:
  • SEC300 or SEC150 buffer for:
o Tethering assays
o Lipid transfer assays
AB
HEPES pH 7.525 mM
NaCl300 / 150 nM
TCEP1 mM

  • SEC150 buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT) for:
o ATG9A proteoliposome (PL) reconstitution
AB
HEPES pH 7.525 mM
NaCl150 mM
TCEP1 mM

Gently mix and sonicate for 00:02:00 in a bath sonicator.
2m
SUV Formation
Pass the rehydrated lipid mixture through a 0.1 µm membrane (Whatman, Cat# 10419504) using a Mini Extruder (Avanti, Cat# 610024).
Final lipid concentration: 1 mg/mL for standard SUVs or 0.5 mg/mL in final suspension.
Lipids Used
AB
LipidSupplier/Details
DOPCAvanti, Cat# 850375C
DOPEAvanti, Cat# 850725C
DOPSAvanti, Cat# 840035C
PI3PSigma-Aldrich, Cat# 850150P-500UG
Liver PISigma-Aldrich, Cat# 840042C-25MG
NBD-PESigma-Aldrich, Cat# 810155P-1MG
Rh-PEInvitrogen, Cat# L-1392
DGS-NTAAvanti, Cat# 790404C
SphingomyelinAvanti, Cat# 860584P-5MG
ATTO390-PEATTO-TEC, Cat# AD 390-161
ATG9A Proteoliposome Reconstitution: Detergent Solubilization
2h
Adjust SUV suspension to 2% CHAPS (Glycon, Cat# D99009-25G).
Mix 100 µL of liposomes with 26 µL of 10% CHAPS (to get final concentration of 2%) and add 4 µL of buffer. Incubate at Room temperature for 01:00:00 .
1h
Add 65 µL of 6xHis-ATG9A-GFP (around 1 micromolar (µM) ) or 65 µL of buffer only to mock samples. Incubate at Room temperature for 01:00:00 .
1h
ATG9A Incorporation
1h
Add purified ATG9A-GFP (in 0.2% DDM) to the CHAPS-solubilized SUVs.
  • Final concentration: 0.5 micromolar (µM) ATG9A-GFP.
  • Use 1:1 volume ratio with SUV-CHAPS mixture.
Add 65 µL of 6xHis-ATG9A-GFP (~ 1 micromolar (µM) ) to 65 µL of CHAPS-solubilized SUVs.
Incubate for 01:00:00 at Room temperature .

1h
Detergent Removal
1h
Dilute the mixture with SEC150 buffer to reduce detergent concentrations:
  • Final DDM concentration ≈ 0.003%
  • Final CHAPS concentration ≈ 0.07%
Perform Overnight dialysis at 4 °C against SEC150 with:
  • 0.1 g/L Bio-Beads SM-2 (Bio-Rad, Cat# 1523920)
1h
Additional Bio-Beads Cleanup
1h
Incubate mixture for 01:00:00 at Room temperature with fresh Bio-Beads.
1h
Removal of Non-Incorporated Material
31m
Centrifuge at low speed in a benchtop centrifuge for 00:01:00 to get rid of Bio-Beads.
1m
Centrifuge at maximum speed in a benchtop centrifuge for 00:30:00 to get rid of aggregates.
30m
Collect supernatant containing ATG9A proteoliposomes for use in downstream assays.