Co-immunoprecipitation: the definition, principle, pros and cos

Posted January 10, 2020 by Bonnibelle

Co-immunoprecipitation (Co-IP) is a classical method for studying protein-protein interactions based on the specific role of antigens and antibodies.

Co-immunoprecipitation (Co-IP) is a classical method for studying protein-protein interactions based on the specific role of antigens and antibodies. The development of co-immunoprecipitation technology has gone through several stages. In the early days, researchers used gel electrophoresis to isolate co-immunoprecipitated proteins: the antigen solution was added to small wells such as agarose, and antiserum was added to adjacent wells. With the antigen and antibody diffusing, large molecules entered in the gel, and an interaction occurs between the two to form a complex. A concentration gradient is formed by diffusion of the antigen and antibody, and a multi-molecular network complex is formed at the optimal concentration. Finally, the large-molecular protein complex is precipitated from the solution. With the development of society, the co-immunoprecipitation technology has been continuously improved. The method of isolating the immune co-precipitation complex is improved to promote the multimer reaction, so that the immune complex is precipitated from the solution.

In the mid-1970s, people began to use solid-phase reactions, using protein A immobilized on the surface of S. aureus to adsorb antibodies, and then bind to the corresponding antigens. With the development of science and technology, this method has been improved to use the surface-immobilized protein A or protein G microspheres to separate antigen-antibody complexes in order to achieve the purpose of detecting antigens or target proteins.

Principle of co-immunoprecipitation

The experimental principle of co-immunoprecipitation is: if X is immunoprecipitated with an antibody to protein X, the protein Y bound to X in the body can also be precipitated. At present, prorein A is often pre-bound and solidified on agarose microbeads and reacted with the solution containing the antigen and the antibody. Prorein A on the agarose microbead specifically binds to the antibody Fc. Due to the specificity of the antibody antigen, protein X is precipitated; There are substances that interact with protein X, and they can precipitate.

Application of co-immunoprecipitation

Determine whether two target proteins are bound in the body
Identify a new role for a particular protein
Isolate and obtain the interaction protein complex in its natural state
With the continuous deepening of protein research, people have combined immunoprecipitation methods with other methods, and based on them, many more complicated technologies have been derived, which makes the analysis methods more diverse and its application range is quite wide. Co-immunoprecipitation is a technique used to study protein-protein interactions and can be applied to the study of protein complexes. It can verify the existence of protein complexes, and then discover new protein complexes. Co-immunoprecipitation technology is combined with immunoblotting or mass spectrometry to determine the binding of bait protein-target protein in its natural state and specific proteins. Co-immunoprecipitation experiments can also be applied to the enrichment and concentration of low-abundance proteins.

At the same time, co-immunoprecipitation is a relatively classic technique for exploring protein-protein interactions. It has a wide range of applications and high credibility in modern medical research. Protein interactions permeate the life activities of every cell in the body. Many phenomena in biology such as replication, transcription, translation, shearing, secretion, cell cycle regulation, signal transmission and intermediate metabolism are all affected by proteins. Some proteins are composed of multiple subunits, and protein-protein interactions are particularly prevalent. Some proteins bind very tightly, while others interact only briefly. However, no matter what kind of situation occurs, they control a large number of events of cell life activity, such as cell proliferation, differentiation and death. And through protein-protein interactions, it can change the dynamic characteristics of intracellular proteins, such as substrate binding properties and catalytic activity. It can also generate new binding sites, which has an effect on changing the specificity of proteins on substrates. Therefore, only by allowing the interaction between proteins to proceed smoothly, the normal life activities of cells can be guaranteed. Because protein-protein interactions are so significant, the study of their detection methods has also received much attention. Since then, the research on protein interrelationships will intensify. In the future, it will not only be confirmed by co-immunoprecipitation technology, but more and more advanced technologies will be worth applying and developing.

The pros and cos


The protein exists in a natural state after modification and translation;
Detects interactions in vitro and in cells;
Antigens and interacting proteins are present at similar concentrations in cells, avoiding human effects caused by overexpression;
It is possible to isolate the interacting protein complex in its natural state.

The sensitivity is not as high as that of affinity chromatography;
High false positive rate, correct control is necessary;
The binding of two proteins may not be directly combined, but a third party may act as a bridge in the middle;
It is necessary to predict what the target protein is before the experiment in order to select the antibody to be detected at last, so if the prediction is incorrect, the experiment will not yield results, and the method itself is risky;
After the protein forms a complex, some epitopes will be masked, which may lead to the use of a pull-down antibody. No matter how the antibody concentration is increased, less than half of the target protein complex can be precipitated. If necessary, it is best to use multiple different antibodies for Co-IP respectively;
Since the natural state is detected, the protein complexes pulled down by Co-IP may be different under different times and different treatments. Of course, as the number of experiments increases, the members of the obtained protein complexes will also bedifferent.
Authenticity of co-immunoprecipitation results

To ensure the authenticity of experimental results in co-immunoprecipitation experiments, the following points should be noted:

Make sure that the co-precipitated protein is obtained by precipitation of the added antibody, not a non-exogenous non-specific protein. Monoclonal antibodies have the advantages of strong specificity, mass production, and easy standardization. The use of monoclonal antibodies can help avoid contamination;
To ensure the specificity of the antibody, if the antibody cannot bind to the antigen in the cell lysate, it will not cause a co-precipitation reaction;
Make sure that protein-protein interactions occur in cells, not because of lysis of the cells.
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Categories Biotech
Last Updated January 10, 2020