Oct 16, 2025
- Tatiana Saccon1,
- Mohan Babu1
- 1Department of Biochemistry, University of Regina
External link: https://doi.org/10.1126/sciadv.adu0726
Protocol Citation: Tatiana Saccon, Mohan Babu 2025. Co-Immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zxzkgqe/v1
Manuscript citation:
Liao S, Kano K, Phanse S, Nguyen M, Margolis E, Fu Y, Meng JX, Moutaoufik MT, Chatterton Z, Saccon T, Broderick K, Aoki H, Simms J, Suteja FX, Sei Y, Huang EJ, McAvoy K, Manfredi G, Halliday G, Babu M, Nakamura K CHCHD2 mutant mice link mitochondrial deficits to PD pathophysiology. Science Advances 11(46). doi: 10.1126/sciadv.adu0726
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: August 20, 2025
Last Modified: October 16, 2025
Protocol Integer ID: 225024
Keywords: ASAPCRN, mitochondria, protein interaction, protein
Abstract
This is a protocol to analyze protein-protein interactions associated with mitochondria using co-immunoprecipitation.
Materials
- RIPA buffer (Thermo Scientific 89900)
- Halt Protease inhibitor cocktail, PIC (Thermo Scientific 78429)
- Primary antibodies
- μMACS Protein A or G magnetic microbeads (Miltenyi Biotec 130-092-945 or 130-092-947)
- μMACS columns (Miltenyi Biotec 130-042-701)
- Magnetic rack
- 2x Laemmli buffer (Bio-Rad 1610737)
Troubleshooting
Procedure
Resuspend 500 µg of mitochondrial lysate in 1 mL RIPA buffer containing a protease inhibitor cocktail (PIC).
Add 3 µL of primary antibody to the lysate and incubate at 4°C with agitation for 1 hour.
Add 100 µL of μMACS Protein A (for rabbit antibodies) or Protein G (for mouse antibodies) magnetic microbeads (Miltenyi).
Incubate at 4°C with agitation for 4 hours.
Pass the microbead-bound protein complexes through μMACS columns.
Wash columns twice with 1 mL of 0.1% RIPA buffer containing PIC.
Wash columns again with 1 mL of detergent-free RIPA buffer.
Elute proteins with 100 µL of 2x Laemmli buffer.
Heat at 95°C before collection.
Proceed to immunoblotting.