Mar 21, 2025

Public workspaceCo-immunoprecipitation V5-NIX

DOI
dx.doi.org/10.17504/protocols.io.q26g7m6qqgwz/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
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DOI: dx.doi.org/10.17504/protocols.io.q26g7m6qqgwz/v1
Protocol Citation: Elias Adriaenssens 2025. Co-immunoprecipitation V5-NIX. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7m6qqgwz/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: January 22, 2025
Last Modified: March 21, 2025
Protocol Integer ID: 119231
Keywords: ASAPCRN
Funders Acknowledgements:
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Abstract
This protocol details co-immunoprecipitation.
Materials
Materials and Reagents

Lysis Buffer:
AB
KCl150 mM
MgCl22.5 mM
Tris-HCl pH 7.420 mM
NP-400.50%
Washing Buffer:
AB
KCl150 mM
MgCl22.5 mM
Tris-HCl pH 7.420 mM

  • V5-NIX (RRID:Addgene_223731)
  • ReagentAnti-V5 agarose beadsMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7345
  • ReagentPierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246
  • ReagentNuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 12-wellThermo FisherCatalog #NP0322BOX


Co-immunoprecipitation
Co-immunoprecipitation
1h
1h
Stably transduce BNIP3/NIX double knockout HeLa cells with lentivirus to express V5-NIX (RRID:Addgene_223731).

After seeding, treat the cells with DFP to induce mitophagy, where indicated.

After the treatment, collect the cells by trypsinization and wash the cell pellet with PBS once before cells were lysed in lysis buffer.

Lysis buffer:
AB
KCl150 mM
MgCl22.5 mM
Tris-HCl pH 7.420 mM
NP-400.50%

Wash
Digestion
Lyse samples for Duration00:20:00 TemperatureOn ice . Then, centrifuge the cell lysates at Centrifigation20000 x g, 4°C, 00:10:00 .

30m
Centrifigation
Temperature
Then, determine the protein concentrations of the cleared protein lysates with the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).
Incubate Amount7 mg of cell lysate DurationOvernight with Amount20 µL of anti-V5 agarose beads (A7345, Sigma-Aldrich).

20m
Incubation
Overnight
In the morning, wash the samples three times in washing buffer and resuspend in protein loading dye, supplement it with Concentration100 millimolar (mM) DTT, and boil it for Duration00:05:00 at Temperature95 °C .

Washing buffer:
AB
KCl150 mM
MgCl22.5 mM
Tris-HCl pH 7.420 mM
5m
Wash
Temperature
Load the samples on 4-12% SDS-PAGE gels (NP0322BOX, Thermo Fisher) and analyze them by western blotting as described above.
Analyze