Co-IP (co-immunoprecipitation) is considered to be one of the classic methods for identifying or confirming protein interaction events in vivo. This method can use antibodies to capture target proteins and their interacting proteins or complexes from samples, and can specifically enrich the target proteins under study. Since non-denaturing conditions are used in the process, the intracellular natural state of the interacting proteins or complexes is retained.
1. Principle of Co-IP Experiment
When cells are lysed under non-denaturing conditions, many protein-protein interactions existing in intact cells are retained. If protein X is immunoprecipitated with an antibody to protein X, proteins Y, Z, etc. that bind to X in vivo can also be enriched. At present, protein A/G is pre-bound and solidified on magnetic beads, and after incubation with a solution containing antigens and antibodies, protein A/G on the magnetic beads can achieve the purpose of enriching and screening and identifying interacting molecules through antibody adsorption of antigens. This method is often used to determine whether two target proteins bind in vivo, and is also used to explore new interaction partners for specific proteins.
2. Experimental Flowchart
3. Experimental steps
3.1 Preparation of immune complexes
3.1.1 Collect cells, add appropriate amount of cell lysis buffer (containing protease inhibitors), lyse on ice for 30 min, centrifuge the cell lysate at 4 °C at maximum speed for 30 min, and take the supernatant; add part of the cell lysate of each sample to a 1.5 mL centrifuge tube together with the recommended amount of IP antibody, and keep the remaining cell lysate as input for downstream Western blot analysis. In addition to the experimental samples, IgG and beads groups can be set as controls.
3.1.2 Dilute the antibody/lysate to 500 μL with immunoprecipitation lysis/washing buffer.
3.1.3 Incubate at room temperature for 1-2 h, or at 4 °C overnight to form immune complexes.
3.2 Co-immunoprecipitation
3.2.1 Add appropriate amount of magnetic beads coupled to protein A/G to a 1.5 mL centrifuge tube.
3.2.2 Add an appropriate amount of immunoprecipitation lysis/wash buffer to the centrifuge tube containing magnetic beads, and gently invert the centrifuge tube several times to mix.
3.2.3 Place the centrifuge tube in a magnetic rack to collect the magnetic beads to one side of the centrifuge tube, and remove the supernatant.
3.2.4 Repeat steps 2-3 2-3 times.
3.2.5 Add the antigen sample/antibody mixture (step A) to the centrifuge tube containing magnetic beads, keep mixing and incubate at room temperature for 1-2 h.
3.2.6 Collect the magnetic beads with a magnetic rack, remove the unbound sample, and save for analysis.
3.2.7 Add an appropriate amount of immunoprecipitation lysis/wash buffer to the centrifuge tube, mix gently, collect the magnetic beads, and discard the supernatant. Repeat the wash 3-5 times.
3.2.8 Low pH elution: Add an appropriate amount of elution buffer to the centrifuge tube. Keep mixing and incubate at room temperature for 10 minutes. Separate the magnetic beads using a magnetic stand and retain the supernatant containing the target protein.
3.2.9 The supernatant containing the target protein can be used for Western blot analysis or protein spectrum identification after adding an appropriate amount of protein electrophoresis loading buffer.
4. Example of experimental results
Co-IP experiments verified the interaction between TssC41 and TssB. RpoA was used as a negative control protein. TssC41 was able to pull down TssB, and conversely, TssB was able to pull down TssC41.
References
[1] Lee C. Coimmunoprecipitation assay. Methods Mol Biol. 2007;362:401-6.
[2] Lin JS, Lai EM. Protein-Protein Interactions: Co-Immunoprecipitation. Methods Mol Biol. 2017;1615:211-219.
[3] Tang Z, Takahashi Y. Analysis of Protein-Protein Interaction by Co-IP in Human Cells. Methods Mol Biol. 2018;1794:289-296.