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Co-Immunoprecipitation (Co-IP)
Protein A agarose beads have physical and chemical properties that enable them to be used in a variety of batch- or column-type affinity purification procedure. Protein A contains four high-affinity binding sites interacting with the Fc region of IgG-class antibodies from selected mammalian species.
In a typical experimental setup, lysates are first prepared from cells or tissue samples that express, either endogenously or exogenously, the protein of interest. Cells are then lysed using a proper lysis buffer, commonly a non-ionic detergent. This lysis step ensures that protein-protein interactions remain intact, by subsequently interrupting lipid-protein or other non-target interactions. The proteins of interest are then captured by incubating the cell lysate with specific antibodies. The resulting immuno-complexes, composed of antibody, the protein of interest (antigen), and antigen-associated proteins, can be precipitated using a beaded resin that is conjugated with an IgG-binding Protein A or G.
Like in IP, the sample then undergoes a series of washes, followed by purification and elution. Tightly bound proteins can also be eluted by boiling the precipitated resins in denaturing Laemmli buffer or by incubating the sample with large amounts of peptides containing the epitope of the antibody. Eluted proteins can be analyzed through similar methods used for IP.
Note: Though the experimental techniques appear similar, the distinction between the two methods lies in that in IP, an antibody is used to purify solely the antigen from a mixture, however in Co-IP an antibody is used to purify its target antigen, along with its binding partners, from a mixed, complex sample.
Experimental Considerations
Experimental conditions must be optimized for each assay, where the lysis buffer and IP antibody are the two most important aspects for a successful experiment. Antibodies must specifically recognize the antigens without interfering with binding partners, which has historically limited the application of co-IP in the detection of complexes between many endogenously expressed proteins.
To overcome this hurdle, the protein of interest and associated proteins are frequently fused with a small peptide sequence, known as an epitope tag (e.g., flag, myc, HA, his, V5) or a fluorescence protein (e.g., GFP, DsRed). Antibodies for these tags that are compatible with IP as well as Co-IP, have been well developed, and are commercially available. As Co-IP purifies and enriches a target protein, it has commonly been combined with secondary studies that target protein-protein interactions, quantification of a protein and its associated proteins, assessing the enzymatic activity of proteins, studying the rate of synthesis or degradation, or determining the molecular weight of a polypeptide.
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Product Ordering Information
Table 1. Protein A-Agarose Resin
| Cat# ▲ ▼ | Product Name ▲ ▼ | Unit Size ▲ ▼ |
| 55000 | Protein A-Agarose Resin | 1 mL |
| 55005 | Protein A-Agarose Resin | 5 mL |