Jan 10, 2026
Co-Immunoprecipitation (co-IP) Protocol
- Ali Ghoochani1,2,3,4,
- Monther Abu-Remaileh1,2,3,4
- 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
- 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
- 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- asap
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Co-Immunoprecipitation (co-IP) Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8z6xl5r/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 13, 2024
Last Modified: January 10, 2026
Protocol Integer ID: 109773
Keywords: protein interaction, studying protein, protocol, cellular model, ip
Abstract
Co-immunoprecipitation (co-IP) is a widely utilized technique for studying protein-protein interactions, particularly in cellular models.
Materials
- 1x PBS (Phosphate-Buffered Saline)
- 1:10 diluted extraction buffer:
- 50 mM HEPES, pH 7.4
- 40 mM NaCl
- 2 mM EDTA
- 1% Triton X-100
- 1.5 mM sodium orthovanadate (NaVO4)
- 30 mM sodium fluoride (NaF)
- 10 mM sodium pyrophosphate (Na4P2O7)
- 10 mM sodium β-glycerophosphate
- Protease inhibitor cocktail
- Anti-Flag magnetic beads (Pierce‱ Anti-DYKDDDDK Magnetic Agarose (Anti-FLAG)(Thermo Fisher Scientific)
- SDS-PAGE materials (Thermo Fisher Scientific)
- Loading dye (5x and 1x)
- Centrifuge
- Shaker
- Incubator at 4°C
- Immunoblotting equipment
Extraction Buffer (1x):
- 50 mM HEPES, pH 7.4
- 40 mM NaCl
- 2 mM EDTA
- 1% Triton X-100
- 1.5 mM Sodium Orthovanadate (NaVO4)
- 30 mM Sodium Fluoride (NaF)
- 10 mM Sodium Pyrophosphate (Na4P2O7)
- 10 mM Sodium β-Glycerophosphate
Troubleshooting
Cell Culture
3d
Seed 15 x 10^6 SH-SY5Y cells expressing the Flag-fusion protein (here: Flag-tagged SLC45A1) and control cells in 15 cm dishes
Incubate the cells for 72:00:00 at 37 °C in a humidified atmosphere with 5% CO₂, or until the cells reach confluence
3d
Cell Lysis
1h
Wash the cells gently with 10 mL 1x PBS to remove any residual media.
Prepare 1 mL of 1:10 diluted extraction buffer supplemented with a protease inhibitor cocktail.
Extraction buffer: Please see "Materials"
Add the extraction buffer and lyse the cells. Use a cell lifter to collect the cells, then transfer the lysate to a new tube.
It is highly recommended to use low-protein-binding tubes for all steps.
Incubate lysate at 4 °C with vortexing for 01:00:00
1h
Centrifuge the lysate at maximum speed for 00:10:00 at 4 °C
10m
Carefully collect the supernatant to new tube
Take 100 µL of the supernatant and add loading dye. Mix well and set aside for later use as the input sample.
Immunoprecipitation
1h
Transfer the remaining 900 µL of supernatant to a new tube containing 25 µL of pre-washed Anti-Flag magnetic beads (Beaded were washed with 1:10 diluted extraction buffer).
Incubate the mixture at 4 °C with gentle rotation overnight. Alternatively, a shorter incubation of 3 hours can also be used if desired
After incubation, wash the beads seven times with 1 ml of 1:10 diluted extraction buffer.
Use a magnet to facilitate the separation of the magnetic beads during the washing process
Important: Change tubes every two washes to ensure effective washing.
After the final wash, add 50 µL of 1x loading dye to the beads. Mix thoroughly.
Alternatively, if non-denatured protein samples are needed, incubate the beads with 200 µg/mL Flag-peptide (prepared in 1:10 diluted extraction buffer 01:00:00 to 02:00:00 at 4 °C
It is recommended to optimize the Flag-peptide concentration and incubation time for your specific Flag-fusion protein
3h