Co-Immunoprecipitation (Co-IP) was developed from the immunoprecipitation technique with which Co-IP shares the fundamental principle of the specific antigen-antibody reaction. Immunoprecipitation(IP) technique is used for isolate individual protein. Using an antibody that is specific for a particular protein, the target protein could be isolated out from a crude lysate of a plant or animal tissue or other biological regent.
Immunoprecipitation of intact protein complexes is known as co-IP, which could pull the entire protein complex out of solution and thereby identify unknown members of the complex. Co-IP is a powerful technique that is used regularly by molecular biologists to analyze protein–protein interactions.
Prepare lysate form cells or tissue samples which express the target protein is the first step of the co-IP. In order to preserve the intact of protein-protein interactions, the lysis buffers should use non-ionic detergents (e.g., NP-40, Triton X-100). Then the target proteins are captured by specific antibodies from total lysate. The resultant immunocomplexes (composed of antibody, protein of interest (antigen), and antigen-associated proteins) can be precipitated using a resin (e.g., agarose, sepharose, or magnetic beads) that is conjugated with IgG-binding Protein A/G. The third step is washes. Irrelevant, non-binding proteins, antigens and any proteins that are bound are eluted by series of washes. Then, the bound proteins which eluted are analyzed by SDS-PAGE/immunoblotting and/or mass spectrometry.
As mentioned above, proper experimental conditions must be determined for each protein-protein interaction. Selection of an optimal lysis buffer and immunoprecipitation antibody are the two most important aspects for the success of a co-IP experiment. To overcome these problem, the protein of interest is often fused with an epitope tag (e.g., flag, myc, HA, his, V5), or a fluorescence protein (e.g., GFP, DsRed) at the terminus of the protein, and ectopically expressed in cells. Antibodies for these “tags” that are compatible with IP/co-IP have been well developed and are commercially available from multiple manufacturers.
Fig1. Schematic of the co-immunoprecipitation procedure.
We will be glad to discuss details of intended interaction studies with you and develop experimental strategies/methods tailored to your requirement. Please see co-IP service in our web and get in contact with Creative BioMart.