Jun 03, 2026
Expression, purification, and characterization of VPS13C
- 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. Expression, purification, and characterization of VPS13C. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz92d4gx1/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 14, 2025
Last Modified: June 03, 2026
Protocol Integer ID: 229726
Keywords: Lipid Transport Protein, VPS13, Protein Purification, characterization of other vps13c mutant, length vps13c from expi293f cell, important for lysosomal membrane damage repair, lysosomal membrane damage repair, lysosome contact site protein, other vps13c mutant, characterization of vps13c vps13c, vps13c vps13c, vps13c, organellar membrane, membrane, length vps13c, expi293f cell, 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 for expressing and purifying the human full-length VPS13C from Expi293F cells as well as structural characterization by negative-stain electron microscopy (EM). Procedures were similarly applied to the expression, purification, and in vitro characterization of other VPS13C mutants.
Guidelines
As the protein yield is low, handle procedures with care.
Materials
Expi293F cells (RRID:CVCL_D615)
Expifectamine (Cat. #A14525, Gibco)
anti-FLAG M2 resin (Cat. #A2220, Millipore-Sigma)
FLAG peptide (Cat. #A6002, Apex Bio)
Safety warnings
Uranyl acetate solution is radioactive and requests special handling on a bench specifically for radioactive materials.
Protein Expression
2d
Culture Expi293F cells in 8% CO2 with orbital shaking at 125 rpm, following the manufacturer’s instructions (Thermo Scientific, MAN0007814).
Transfect 200 mL Expi293F cells at 2.4–2.8 × 10^6 cells/mL with 200 μg plasmid encoding full-length VPS13C or mutant constructs using Expifectamine. Add the manufacturer-supplied enhancers 18 h after transfection.
Harvest cells 48 h after transfection, flash-freeze in liquid nitrogen, and store at −80°C.
Protein Purification
6h 30m
Thaw cells at room temperature and resuspend in 20 mL buffer A (500 mM NaCl, 50 mM HEPES pH 7.8, 10% glycerol, 1 mM TCEP) supplemented with 1× protease inhibitor cocktail.
30m
Lyse cells by three freeze–thaw cycles between liquid nitrogen and a room-temperature water bath, then homogenize with a Dounce homogenizer.
1h
Clarify the lysate by centrifugation at 27,143 × g for 30 min in a JA-20 rotor.
30m
Incubate the supernatant with 100 μL anti-FLAG M2 resin for 2 h at 4°C.
2h
Wash the resin twice with 15 mL buffer A, then incubate the resin overnight in buffer A supplemented with 1 mM freshly made ATP and 2 mM MgCl2 to remove chaperones.4 °C Overnight
Wash the resin twice more with buffer A and elute bound protein with buffer A containing 0.25 mg/mL FLAG peptide in five sequential 100 μL incubations of 20–30 min each. Pool the eluates.
Purified VPS13C-3xFLAG
30m
Load the pooled eluates onto a Superose 6 10/300 column pre-equilibrated with buffer B (200 mM NaCl, 50 mM HEPES pH 7.2, 6% glycerol, 1 mM TCEP).
For structural analysis of full-length VPS13C, perform size-exclusion chromatography in buffer C (200 mM NaCl, 50 mM HEPES pH 7.2, 1 mM TCEP), then collect and concentrate peak fractions.
2h
Negative-Stain Characterization
1h 48m
Glow-discharge 400-mesh carbon-coated copper grids for 35 s at 25 mA.
10m
Apply 5–7 μL protein sample diluted to 50-100 nanomolar (nM) to the grid and incubate for 30 s.
3m
Blot the sample and stain with three to five sequential 5 μL drops of 2% uranyl acetate, allowing the final drop to incubate for 30 s before blotting to dryness.
5m
Acquire images on an FEI Tecnai T12 transmission electron microscope at 120 kV and 52,000× nominal magnification, corresponding to 2.14 Å/pixel at the specimen level.
Representative negative stain EM image
30m
Perform particle picking and 2D classification in CryoSPARC.
1h