Immunoprecipitation

When conducting immunoprecipitation, antibodies are bound on microbeads. Most often, agarose microbeads are used . Microgranules having magnetic properties can also be used. Using a magnet, magnetic microbeads carrying can be kept in a test tube while removing components of the sample that are not bound to the antibody ^[1] .

Three technologies of immunoprecipitation are distinguished depending on the method of binding antibodies on microbeads. In classical technology, microbeads coated with protein A or protein G are used . Protein A, like protein G, can bind to the Fc region of a wide range of antibodies. The antibody specific for the secreted protein is incubated with the mixture from which the protein is to be isolated. After the complex of the secreted protein ( antigen ) with the antibody is formed, the microgranules coated with protein A or protein G are introduced into the mixture. The antigen-antibody complexes bind on the microgranules. By centrifugation and washing, microgranules with bound antigen-antibody complexes are separated from the mixture. Antigen and antibody elute from the microbeads. Sometimes microgranules with which antibodies have previously been bound are introduced into the mixture. The main disadvantage of the classical technology is that when the protein is eluted from the microparticle, the antibody will also be removed from the microparticle. As a result, the secreted protein will be contaminated and antibodies cannot be reused ^[1] .

Contamination of the secreted protein and loss of antibodies can be avoided by immobilizing the antibodies on the microparticle. There are two technologies: immobilization due to covalent crosslinking of the antibody and protein A or protein G located on the surface of the microparticle, and immobilization due to the formation of a covalent bond between the antibody and the microparticle material. These technologies also have their drawbacks. The disadvantage of covalent crosslinking of an antibody and protein A or protein G is that the crosslinking reagent can form covalent bonds in arbitrary portions of the antibody, damaging the active center , and, as a result, the antibody loses its ability to bind antigen. The disadvantage of covalent crosslinking of the antibody and the microparticle material is that, unlike technologies using protein A or protein G, an arbitrary region of the antibody will bind to the microparticle, which can lead to a loss of the ability of the antibody to bind antigen ^[1] .

In a situation where antibodies to the secreted protein are not available, a tag (for example, the ) can be attached to it using the genetic engineering methods ^[2] .

The advantages of immunoprecipitation include the fact that in this method antigens interact with antibodies in their for further separation and quantitative analysis. Moreover, the immunoprecipitation method allows protein concentration. The disadvantage of this method is that for effective detection, the protein must be radioactively labeled ^[3] .

Co-immunoprecipitation

Co-immunoprecipitation (Co-IP) is the immunoprecipitation of a whole protein complex , which is based on the use of an antibody specific for one of the proteins that make up the complex. By binding this protein, the antibody binds the entire complex. As a result, it becomes possible to identify all the proteins that make up the complex ^[1] .

Chromatin Immunoprecipitation

(ChIP) is a method of finding the binding sites of the studied in the genome . For this, DNA is isolated from the cell and cut into small fragments, and then immunoprecipitation is carried out using antibodies to the studied DNA-binding protein. As a result, complexes consisting of the studied DNA-binding protein and a DNA fragment bind to antibodies. Such DNA fragments are the binding sites of the studied DNA-binding protein in the genome. To read the nucleotide sequence of these DNA fragments, DNA microarrays (ChIP-on-chip) or modern sequencing methods (ChIP-seq) are used ^{[4] [5]} .

RNA Immunoprecipitation

RNA immunoprecipitation (RIP) is a method that allows you to identify RNA molecules that interact with the studied RNA-binding protein, and to determine the binding sites of the studied protein with RNA molecules. The RNA immunoprecipitation procedure is similar to chromatin immunoprecipitation. To read the nucleotide sequence of the isolated RNA fragments, DNA microarrays are used, having previously obtained complementary DNA fragments (RIP-chip), or modern sequencing methods (RIP-seq) ^[6] .

• B. Alberts, A. Johnson, D. Lewis et al. Molecular cell biology / Translated from English by A. N. Dyakonova, A. V. Duba, and A. A. Svetlova. Ed. E. S. Shilova, B. P. Kopnin, M. A. Lagarkova, D. V. Kuprash .. - M. — Izhevsk: Research Center “Regular and chaotic dynamics”, 2013. - V. 1. - P. 663 -665. - 1052 s. - ISBN 978-5-4344-0137-1 .

• Protocols and technical data on immunoprecipitation on the ThermoScientific website (neopr.) .
• Immunoprecipitation Protocols on Cell Signalling Technology (neopr.) .