Our new pocket guide contains a set of steps to help you with your experimental design.Download or request a copy
Learn about the conflicting nature of R-loops, a trending area of cancer research.Learn more
Be the first to know when we launch new products and resources to help you achieve more in the lab.Sign up now
In an immunoprecipitation (IP) experiment, an antibody is cross-linked to agarose, sepharose or magnetic beads in order to capture a protein of interest present in a lysate. The technique is mainly used for the analysis of protein-protein interactions, the characterization of protein complexes and the identification of post-translational modifications. For these purposes IP samples are first run on SDS-PAGE gels, followed by transfer onto membranes for western blot analysis. Alternatively, gels are silver or Coomassie stained to visualize the immunoprecipitated protein(s). Bands of interest are then extracted from the gel and prepared for mass-spectrometrical analysis. With the help of this technique, both new interaction partners and modifications, such as phosphorylated residues, may be determined.
When the identification of new interaction partners or entire protein complexes is desired, the IP procedure is referred to as co-immunoprecipitation (co-IP). The name originates from the aspiration to not only immunoprecipitate the protein against which the antibody cross-linked to beads was raised but to also co-immunoprecipitate other proteins. However, to confirm a protein-protein interaction the recommendation is to perform the co-IP experiment both ways. For this purpose, after having identified a new interaction partner, this protein in return should be immunoprecipitated. If that IP pulls-down the protein initially used for the co-IP the interaction is verified. However, this verification does not mean that the two proteins interact directly as the interaction could be mediated by a third protein, such as an adaptor protein.
When designing an IP experiment it is important to select an antibody that has been tested in IP. In contrast to other applications, antibodies used in IP have to recognize native rather than denatured proteins. Depending on the protein structure and folding, certain epitopes may be buried and are therefore not accessible to antibodies. When no crystal structure is available the conformation of a native protein, especially when part of a larger complex, is difficult to predict. Based on this difficulty in predetermining which epitopes might be exposed, polyclonal antibodies are often favored when first establishing an IP protocol; their poly-specific nature increases the likelihood that one antibody present in the mixture recognizes an exposed epitope.
To minimize the use of different antibodies and the need to identify an antibody recognizing the native protein, proteins of interest are often tagged to fluorescent proteins or epitope tags. These tagged proteins are either expressed at physiological levels in a knock-in context or at higher levels when the protein is over-expressed.
The host, and in the case of a monoclonal antibody, the isotype play an important role in determining whether the use of protein A or protein G agarose, sepharose or magnetic beads is advisable. Binding affinities to protein A and protein G vary significantly between species and isotypes. For example, rat IgG2b has no affinity for protein A while rabbit IgG has a stronger affinity for protein A than protein G. For an overview of antibody binding affinities, please click here.