Jun 02, 2026
Endogenous calmodulin co-purified with VPS13s detected by western blotting
- 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. Endogenous calmodulin co-purified with VPS13s detected by western blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31w17l25/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: 230220
Keywords: Lipid transport protein, VPS13, western blot, lysosome contact site protein, vps13 from chaetomium thermophilum, important for lysosomal membrane damage repair, lysosomal membrane damage repair, endogenous calmodulin co, organellar membrane, purified with vps13, calmodulin, membrane, chaetomium thermophilum, uncovered novel regulatory mechanism, western blotting vps13c, vps13c, lipid, novel regulatory mechanism, vps13
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 describes how we see calmodulin consistently bind to VPS13C and VPS13 from Chaetomium thermophilum by western blotting.
Materials
anti-calmodulin antibody (Cat. #HY-P82082, MCE)
goat anti-rabbit HRP conjugate (Cat. #AP307P, Sigma Aldrich)
ECL substrates (Cat. #32106, Thermo Scientific)
Protocol materials
CalmodulinMedChemExpressCatalog #HY-P82082
Goat Anti-Rabbit IgG Antibody, (H+L) HRP conjugateMilliporeSigmaCatalog #AP307P
The purification of VPS13s
8h
Following the expression and purification protocol (doi.org/10.17504/protocols.io.rm7vz92d4gx1/v1), either C-terminally tagged VPS13C or N-terminally tagged CtVPS13 was expressed in Expi293F cells.
6h
Proteins were purified in buffer B (200 mM NaCl, 50 mM HEPES, pH 7.2, 6% glycerol, 1 mM TCEP) supplemented with either Ca2+ or EGTA.
2h
Western blot analysis of co-purified calmodulin
19h 30m
For better visualization of calmodulin, resolve the eluted proteins on 4–20% Tris-glycine precast gels.
For better visualization of VPS13s, resolve the eluted proteins on 3-8% Tris-acetate precast gels.
1h
Transfer proteins to PVDF membranes at 90 V for 01:30:00 at 4 °C in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol).
2h
Block membranes for 01:00:00 at Room temperature in 3% BSA in TBST.
1h
Incubate membranes Overnight at 4 °C with anti-calmodulin primary antibodyCalmodulinMedChemExpressCatalog #HY-P82082 diluted 1:1000 in 3% BSA/TBST.
12h
Wash membranes three times with TBST, each for 00:15:00 .
1h
Incubate membranes for 01:00:00 at Room temperature with goat anti-rabbit HRP-conjugated secondary antibodyGoat Anti-Rabbit IgG Antibody, (H+L) HRP conjugateMilliporeSigmaCatalog #AP307P diluted 1:1000 in TBST.
1h
Wash membranes three times with TBST, each for 00:15:00 .
1h
Develop membranes with ECL substrate and image using an ImageQuant LAS 4000.
30m