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Co-Immunoprecipitation
Introduction of Co-Immunoprecipitation (Co-IP)
Co-immunoprecipitation (Co-IP) technology utilizes the specificity of antibodies to recognize antigens, selects an antibody that has already targeted a protein, and pulls the entire protein complex out of the solution by targeting this protein, thereby identifying unknown proteins in the complex. This method requires tight protein binding. After elution, gel electrophoresis separation, combined with Western Blot and other techniques, quantitative or qualitative analysis of the content of specific antigens in the sample. Due to the use of non-denaturing conditions during the process, the physiological interactions between proteins and peptides, as well as between proteins within intact cells, were preserved. The validation of the binding of two proteins in cells is consistent with the actual binding situation in vivo, and the reliability of the detection results is high. It is often used to study protein-peptide and protein-protein interactions.
Principle of Co-Immunoprecipitation (Co-IP)
After cell lysis, the interactions between proteins are preserved. The antibody of protein X binds to protein A/G on agarose magnetic beads. When the antibody immunoprecipitates antigen protein X, the target protein Y that can bind to antigen protein X is also precipitated, forming an immune complex of "target protein+antigen protein+antibody+protein A/G". The target protein is detected by Western Blot or mass spectrometry [2].
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Figure 1: Schematic diagram of immunoprecipitation (Co IP)
Advantages of Co-Immunoprecipitation (Co-IP)
(1) Proteins exist in a post-translational modified state.
(2) Western Blot can be used to detect interactions between known proteins, and mass spectrometry can be used to identify interactions between known and unknown proteins.
(3) Multiple samples can be selected for experiments to verify the natural binding of two proteins in cells, which is in line with the actual situation in vivo and has high reliability in the detection results.
(4) Immunoprecipitation can detect protein-protein interactions in a natural state [5], and this method can minimize human influence as much as possible.
Precautions for immunoprecipitation (Co IP)
(1) Specific IP level bait protein antibodies are required (emphasis);
(2) If there is no specific antibody, it is necessary to consider constructing a recombinant tag plasmid and conducting Co IP experiments with the tag antibody;
(3) Experimental risks of sample types: overexpression of cell samples
(4) CoIP MS risk: Due to the presence of high abundance proteins with specific antibodies, mass spectrometry may not be able to identify bait proteins.
(5) The use of monoclonal antibodies can prevent contamination and ensure that the protein precipitates due to the action of the antibodies.
(6) Antibodies need to have specificity and ensure that they can bind to antigens in the sample.
Reference
[1] Whiterhiannon R , Ponsfordamy H , Weekesmichael P ,et al.Co-immuno-precipitation (co-IP) and yeast-2-hybrid (Y2H) analyses of TsUBE2L3.[J].PLOS Pathogens, 2016.DOI:10.1371/journal.ppat.1005977.g004.
[2] Tiwari K K . Study of nitric oxide synthase-interacting proteins by mammalian Co-IP and yeast two-hybrid assay[J].Dissertations & Theses - Gradworks, 2012.
[3] Steinbrenner J , Eldridge M ,Daniel F. A. Tomé, et al.A Simple and Fast Protocol for the Protein Complex Immunoprecipitation (Co-IP) of Effector: Host Protein Complexes[J].Methods in Molecular Biology, 2014, 1127:195.DOI:10.1007/978-1-62703-986-4_16.
[4] Hui, Hu, Tao, et al.Screening and identification of proteins interacting with IL-24 by the yeast two-hybrid screen, Co-IP, and FRET assays.[J]. Anti-cancer drugs, 2016.DOI:10.1097/CAD.0000000000000343.
[5] Steinbrenner J , Eldridge M ,Daniel F. A. Tomé, et al.A Simple and Fast Protocol for the Protein Complex Immunoprecipitation (Co-IP) of Effector: Host Protein Complexes[J].Methods in Molecular Biology, 2014, 1127:195.DOI:10.1007/978-1-62703-986-4_16.
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