Jun 02, 2026
Co-floatation assay of VPS13C with SUVs
- Dazhi Li1,2,
- Karin Reinisch1,2
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Dazhi Li, Karin Reinisch 2026. Co-floatation assay of VPS13C with SUVs. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n9nqlwz/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: October 19, 2025
Last Modified: June 02, 2026
Protocol Integer ID: 230231
Keywords: Lipid Transport Protein, VPS13, Liposome floatation assay, important for lysosomal membrane damage repair, lysosome contact site protein, lysosomal membrane damage repair, rab7 on the membrane, membrane, small unilamellar vesicle, organellar membrane, vps13c, suvs vps13c, lipid, interaction between the vab domain, floatation assay, atg2-c region, uncovered novel regulatory mechanism
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: Grant ID: ASAP-000580
Abstract
VPS13C is a ER-lysosome contact site protein that is thought to transport lipids between the two organellar membranes and important for lysosomal membrane damage repair. We employed structure-function analysis of purified VPS13C and uncovered novel regulatory mechanisms. This protocol details the floatation assay to characterize VPS13C's binding to small unilamellar vesicles (SUVs), where we discovered VPS13C's binding to membrane depends on both its ATG2_C region and the interaction between the VAB domain and Rab7 on the membrane.
Guidelines
Proteins and liposomes are stored in 4°C for no more than a week.
Materials
POPC (Cat. #850457)
POPE (Cat. #850757)
DGS-NTA (Ni) (Cat. #790404)
Lissamine Rhodamine PE (Rhod-PE; Cat. #810150)
OptiPrep (Cat. #D1556, Sigma Aldrich)
Protocol materials
Rhod-PEAvanti Polar Lipids, Inc.Catalog #810150
16:0-18:1 PC (POPC) (format: Chloroform)Avanti Polar LipidsCatalog #850457C
16:0-18:1 PEAvanti Polar Lipids, Inc.Catalog #850757
18:1 DGS-NTA(Ni) (format: Chloroform)Avanti Polar LipidsCatalog #790404C
Preparation of SUVs
3h 10m
Mix 16:0-18:1 PC (POPC) (format: Chloroform)Avanti Polar LipidsCatalog #850457C , 16:0-18:1 PEAvanti Polar Lipids, Inc.Catalog #850757 , 18:1 DGS-NTA(Ni) (format: Chloroform)Avanti Polar LipidsCatalog #790404C , and Rhod-PEAvanti Polar Lipids, Inc.Catalog #810150 at 84.8:10:5:0.2 mol%.
1h
Dry the lipid mixture under a gentle nitrogen stream and desiccate under vacuum overnight.
20m
Rehydrate the dried lipid film in buffer D (200 mM NaCl, 25 mM HEPES, pH 7.5, 1 mM TCEP) to a final lipid concentration of 10 millimolar (mM) .
Incubate the suspension at 37 °C for 01:00:00 , mixing gently every 00:20:00 .
1h
Subject the suspension to five freeze–thaw cycles.
20m
Extrude the liposomes 31 times through a 200 nm polycarbonate filter using the Avanti Mini-Extruder to generate SUVs.
30m
Flotation assay of SUVs
4h 50m
In a 100 μL reaction, mix 1–5 μg purified full-length VPS13C or VPS13C-ΔATG2C with 1 mM SUVs.
10m
Add equal mass of Rab7A(Q67L/T22N)-6xHis or 6xHis-ATG7 as a negative control, together with 1 mM GTP and 2 mM Mg2+, and incubate at room temperature for 00:20:00 .
30m
For the flotation assay, mix 90 μL of the reaction with an equal volume of 60% OptiPrep (Cat. #D1556, Sigma Aldrich) to generate a 30% layer.
20m
Transfer the sample to a 0.8 mL ultracentrifuge tube (Cat. #344090, Beckman), then overlay with 300 μL of 20% OptiPrep and 100 μL reconstitution buffer.
10m
Centrifuge at 193,911 × g for 01:00:00 in an SW-55 Ti rotor 18 °C .
1h
Collect 100 μL from the top fraction containing floated SUVs.
10m
Analyze a 10 μL input sample and 30% of the top fraction by SDS-PAGE on 3–8% Tris-acetate precast gels.
2h
Quantify band intensities in ImageJ and calculate floated protein as the normalized top fraction divided by the input, shown as a percentage.
30m