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Co-IP experiment
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Co-IP experiment

Experimental Principle

 
When cells are lysed under non-denaturing conditions, many protein–protein interactions that exist within intact cells are preserved. If antibody against protein A is used to immunoprecipitate A, then protein B, which binds to A in vivo, can also be precipitated. The resulting complex is subsequently detected by Western blotting (WB).
 

Experimental Procedure

 

1. Culture the cells required for the experiment

2. Exogenous CO-IP requires transfection with an exogenous plasmid.

3. Cell lysis (RIPA buffer is commonly used, with PMSF and other protease inhibitors added)

 

4. Immunoprecipitation reaction

 

① Magnetic bead pre‑processing (using protein A/G‑ or HA‑tagged magnetic beads)

② The target antibody is conjugated to magnetic beads (typically at an antibody concentration of 10–50 µg/mL).

③ Antigen precipitation assay (binding of antibody-conjugated magnetic beads to cell lysate)

④Antigen elution (both denaturing and non-denaturing types)

⑤WB Detection and Result Analysis
 

Experimental Precautions

 

1. Before use, the magnetic beads should be thoroughly shaken to ensure uniformity.
2. Select antibodies with high specificity for the immunoprecipitation assay.
3. The target protein and its interacting proteins must not be expressed at excessively low levels.
4. Operate under appropriate experimental conditions to prevent instability in the experimental data.
5. Use of control antibodies: Monoclonal antibody: normal mouse IgG or another type of monoclonal antibody; Rabbit polyclonal antibody: normal rabbit IgG.
6. Cell lysis is performed under mild conditions to avoid disrupting all intracellular protein–protein interactions; non-ionic detergents such as NP‑40 or Triton X‑100 are commonly used. Various enzyme inhibitors, including the commercially available protease inhibitor PMSF, should be added to the cell lysate.

 

CO-IP experimental results