Jun 03, 2026
Complex formation between VPS13C and Rab7
- 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. Complex formation between VPS13C and Rab7. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly4q4qlx9/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 20, 2025
Last Modified: June 03, 2026
Protocol Integer ID: 230223
Keywords: Lipid Transport Protein, VPS13, Rab7, lysosome contact site protein, important for lysosomal membrane damage repair, lysosomal membrane damage repair, rab7 vps13c, complex formation between vps13c, rab7 complex, organellar membrane, membrane, vps13c, rab7, purifying vps13c, uncovered novel regulatory mechanism, lipid, novel regulatory mechanism
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: ASAP-000580
NIH
Grant ID: R35GM131715
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 method to isolate and detect a VPS13C-Rab7 complex formed by pull-down assay and the method to directly form a complex by crosslinking purifying VPS13C and Rab7.
Materials
GTP (Cat. # G8877, Sigma Aldrich)
Strep-Tactin XT 4Flow high-capacity resin (Cat. #2-5030-002, IBA)
D-Biotin (Cat. #BG-00, G-Biosciences)
Amylose resin (Cat. #E8021, NEB)
maltose (Cat. #M5895, Sigma Aldrich)
Protocol materials
Sodium GTPMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8877
Co-immunoprecipitation of VPS13C by Strep-Rab7A from Expi293F cells
13h
Co-express N-terminally Strep-tagged Rab7A and C-terminally 3xFLAG-tagged VPS13C full-length or mutant constructs in Expi293F cells. For transfection, mix 30 μg Rab7A plasmid and 70 μg VPS13C plasmid in Opti-MEM and Expifectamine and transfect into 100 mL Expi293F cells.
1h
After 48 h, harvest cells and resuspend in 10 mL buffer A (500 mM NaCl, 50 mM HEPES, pH 7.8, 10% glycerol, 1 mM TCEP) supplemented with 1 mM GTP Sodium GTPMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8877 and 2 mM MgCl2.
30m
Lyse cells by three freeze–thaw cycles and clarify the lysate.
1h
Divide the clarified lysate, using 30% for FLAG pull-down with 50 μL anti-FLAG M2 resin and 70% for Strep pull-down with 50 μL Strep-Tactin XT 4Flow high-capacity resin. Incubate each pull-down for 02:00:00 at 4 °C .
2h
Wash the resin four times with 15 mL buffer A.
30m
Elute bound proteins three times in 50 μL buffer A supplemented with 0.5 mg/mL FLAG peptide for FLAG pull-down or 50 mM D-biotin for Strep pull-down, incubating for 00:20:00 for each elution.
1h
Pool the eluates and concentrate to a final volume of 50 μL.
30m
Analyze Strep pull-down eluates by SDS-PAGE. For better separation, resolve Rab7A on 4–20% Tris-glycine gels and co-purified VPS13C on 3–8% Tris-acetate gels.
2h
Quantify VPS13C binding by normalizing the intensity of each co-purified VPS13C band to the corresponding Rab7A band, then normalize to the full-length VPS13C control.
30m
For the VPS13C-VAB domain alone, detect co-purified protein by anti-FLAG western blot because it is not reliably quantified by Coomassie staining.
4h
Cross-linking of independently purified VPS13C and Rab7A
10h 30m
Transfect and express MBP-TEV-Rab7A-6xHis in Expi293F cells. Lyse cells by three freeze–thaw cycles and clarify the lysate.
1h
Incubate clarified lysate with Amylose resin for 02:00:00 at 4 °C , wash the resin, and elute bound protein with buffer A supplemented with 50 mM maltose.
2h
Run pooled eluates on a Superose 6 size-exclusion column equilibrated in buffer B (200 mM NaCl, 50 mM HEPES, pH 7.2, 6% glycerol, 1 mM TCEP) to remove maltose.
2h
Mix purified full-length VPS13C-3xFLAG and MBP-Rab7A-6xHis at a 1:10 molar ratio and incubate Overnight at 4 °C .
Add 0.1% glutaraldehyde to VPS13C alone, Rab7A alone, or the VPS13C+Rab7A mixture, and incubate for 00:15:00 at Room temperature .
20m
Quench the cross-linking reaction with 100 mM Tris, pH 8.0, for 00:10:00 at Room temperature .
10m
Subject all samples to MBP pull-down as described above.
3h
Analyze cross-linked VPS13C–Rab7A complexes by SDS-PAGE.
2h