Immunoprecipitation refers to a method of preliminarily separating the target protein by precipitating the antigen (usually the target protein) from the mixed system by using the characteristic that the antibody can specifically bind to the antigen.
Coimmunoprecipitation is a method to detect the existence of specific interactions between two protein molecules in vitro. The principle is very simple. If two proteins can interact specifically in an in vitro system, then when an antibody against one protein is used for immunoprecipitation, the other protein will also be precipitated at the same time. Different from yeast two-hybrid technology, co-immunoprecipitation technology utilizes the immune reaction between antigen and antibody, which is a method based on the study of protein-protein interaction in a non-cellular environment in vitro.
It is not difficult to see that the principles and methods used in co-immunoprecipitation and immunoprecipitation techniques are roughly similar, except that in co-immunoprecipitation, the binding and precipitation of the target protein is replaced by another protein that interacts with it. On the basis of co-immunoprecipitation or immunoprecipitation, by further combining with other techniques, such as polyacrylamide gel electrophoresis, the molecular weight and other characteristics of the target protein can be further identified.
The selection of antibodies
Polyclonal antibodies are the most widely used because they are relatively simple to prepare, can bind to multiple sites of the target protein molecule, and the formed antigen-antibody complexes are relatively stable. However, the disadvantage of polyclonal antibodies is that there are many non-specific bindings, which often lead to increased background and certain false positive results.
Compared with polyclonal antibodies, monoclonal antibodies often only bind to one epitope and have a single binding specificity, so there is less chance of non-specific binding, and can be used to determine the special structure of a certain part of the target protein, and even Can be used to distinguish different forms of the same target protein such as conformational changes and modifications. But conversely, the property of monoclonal antibodies to only bind to a single epitope can also cause cross-reactivity between different target proteins with the same epitope.
For immunoprecipitation and co-immunoprecipitation, the force between the antibody and the target protein is preferably just enough to achieve the effect of separation. The binding force is too weak to achieve the purpose of separation; conversely, the binding force is too strong and subsequent purification is not used. Therefore, for monoclonal antibodies, its affinity with the target protein is particularly important. Generally, antibodies with avidity less than 10 7 M – 1 are not suitable for immunoprecipitation.
The ideal antibody for immunoprecipitation and co-immunoprecipitation should be a complex of monoclonal antibodies directed against different epitopes on the same target protein molecule. At the same time, in order to ensure the separation effect, the antibodies are often fixed on a solid support such as polysaccharide gel particles Sepharose CL-4B in advance, or the antibodies are combined with unrelated anti-immunoglobulin antibodies and proteins that can bind to immunoglobulins. A or protein G linkage.
The target protein for immunoprecipitation is generally obtained from cell lysates and can be either isotopically labeled or unlabeled. If it is the former, after immunoprecipitation and then polyacrylamide gel electrophoresis, the presence of the target protein can be detected by pressing the tablet; if it is the latter, after immunoprecipitation and polyacrylamide gel electrophoresis, silver identification by staining or immunoblotting.
Required reagents and solutions
Modified RIPA solution (cell lysis solution), dilution buffer (usually PBS solution), TSA solution, 0.05mol/l Tris (pH6.8), 2×SDS protein loading buffer, antibody and Sepharose or anti-immunoglobulin antibody , protein A, protein G cross-linker.
Composition of modified RIPA solution (100ml system)
Tris-HCl: 50 mM, pH 7.4
Sodium deoxycholate: 0.25%
EDTA: 1 mM
Aprotinin, leupeptin, aprotinin: 1µg/ml each
Na 3 VO 4 : 1 mM
NaF: 1 m