Co

The topic of co-immunoprecipitation (co-IP) is best preceded by an overview of immunoprecipitation (IP) to help frame an understanding of the principles involved. The description of IP methodology here is brief.

Immunoprecipitation is one of the most widely used methods for antigen detection and purification. The principle of an IP is very straightforward: an antibody (monoclonal or polyclonal) against a specific target protein forms an immune complex with that target in a sample, such as a cell lysate. The immune complex is then captured, or precipitated, on a beaded support to which an antibody-binding protein is immobilized (such as Protein A or G), and any proteins not precipitated on the beads are washed away. Finally, the antigen (and antibody, if it is not covalently attached to the beads and/or when using denaturing buffers) is eluted from the support and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), often followed by western blot detection to verify the identity of the antigen.

Schematic summary of a standard immunoprecipitation assay.

Co-immunoprecipitation is an extension of IP that is based on the potential of IP reactions to capture and purify the primary target (i.e., the antigen) as well as other macromolecules that are bound to the target by native interactions in the sample solution. Therefore, whether or not an experiment is called an IP or co-IP depends on whether the focus of the experiment is the primary target (antigen) or secondary targets (interacting proteins).

Schematic summary of a standard co-immunoprecipitation assay.

While the co-IP methodology is straightforward, performing a co-IP reaction and identifying physiological protein–protein interactions can be difficult because of the nature of the interaction, nonspecific binding to IP components and antibody contamination that may mask detection. The following sections describe each aspect of the co-IP approach that can be optimized to improve detection.

Because co-immunoprecipitation depends so much on protein–protein interactions in order to detect the bound proteins, the ability to maintain stable physiological interactions throughout the mechanical and chemical stresses of the incubation and washing steps is a critical factor when performing a co-IP reaction. Therefore, low-affinity or transient protein–protein interactions may not be detected by co-IP unless the interaction can be stabilized.

A key factor in maintaining complex formation throughout the steps required for co-IP is the lysis and wash buffers. Many protein interactions will remain intact after lysis using standard non-denaturing lysis buffers, as described in the Immunoprecipitation method in the Pierce Protein Methods library. Buffers with low ionic strength (i.e., Protein Preparation Handbook

Learn more about how to desalt, buffer exchange, concentrate, and/or remove contaminants from protein samples, immunoprecipitation and other protein purification and clean up methods using various Thermo Scientific protein biology tools in this 32-page handbook.

One of the most commonly encountered problems with both IP and co-IP approaches is interference from antibody bands during gel analysis. In those cases where several proteins may be co-precipitated with the target, the presence of the co-eluted antibody light and heavy chains (25- and 50-kDa bands in reducing SDS-PAGE gels, respectively) in the sample can obscure the results. The ideal situation would be to analyze the co-IP without contamination of the eluted antigen with antibody; with this potential interference eliminated, only the co-precipitated proteins would be present and detected on a gel.

Antibody contamination can be circumvented using methods described in the Overview of Immunoprecipitation Methods page, including crosslinking antibody to Protein A/G–coated beads or covalently binding antibody directly to treated beads. An added benefit of these approaches is the potential reuse of the antibody-coated beads. A key to preventing antibody contamination using these strategies is to elute the antigen under non-denaturing conditions; otherwise, the denatured antibody fragments will be eluted with the antigen.

Another direct coupling approach incorporates the binding association between streptavidin and biotin, in which the IP antibody is biotinylated and the beads are coated with streptavidin. The immune complexes are captured by the beads, and because biotin binds strongly to streptavidin, the antibody is not eluted from the beads when mild conditions are used to release the target antigen. A wide selection of affinity resins, magnetic beads and coated plates based on immobilized avidin, streptavidin or Thermo Scientific NeutrAvidin Protein facilitates this strategy.

By contrast, when popular fusion tags are incorporated into the primary target protein to be used in a co-IP experiment, pre-immobilized anti-fusion tag antibodies may be used for protein complex purification. For example, antibodies specific to the HA (YPYDVPDYA) or c-Myc tag (EQKLISEEDL) can be covalently immobilized to beaded agarose resin, enabling their use in IP or co-IP experiments involving HA- or c-Myc-tagged "bait" proteins.

Co-IP of active Rac1 with HA-tagged Pak1-PBD (p21 binding domain). Human 293 cells were transfected with HA-Pak1 protein binding domain (PBD) alone or co-transfected with constitutively activate Rac1 (Q61L). Anti-HA agarose slurry (6 µL) was incubated with 50 µL HA-tagged positive control lysate (Lane 1) or 500 µL cell lysate from Rac1 (Q61L) and HA-Pak-PBD co-transfected cells (Lane 2). HA-Pak1-PBD-transfected cells (Lane 3) or non-transfected cells (Lane 4). IP and co-IP reactions were performed at 4°C overnight. The western blot was first probed with anti-Rac1 antibody (A) and then reprobed with anti-HA antibody (B).

When a protein–protein interaction is detected, it is critical to confirm that the detection is a true physiological interaction as opposed to an artifactual interaction due to some aspect of the protocol. A summary of approaches to verify a protein–protein interaction follows.

The quality and specificity of antibodies range from those that weakly bind and are nonspecific to those that show high affinity and specificity for a single epitope. A critical part of confirming any detected protein–protein interaction is first confirming that the target protein can be immunoprecipitated from the sample, which is confirmed using well-characterized antibodies that are known to specifically bind to the target antigen. If data on the specificity of an antibody is not available, then cells that lack the target protein should be used with the IP antibody to show that nothing is precipitated using the antibody. Of course, when testing non-characterized antibodies, one should always include a control to show that the target protein can be precipitated from a stock of purified protein using the test antibody.

Whereas a number of antibody validation strategies can be used to verify the specificity of an antibody, antibody validation by immunoprecipitation followed by mass spectrometry analysis (IP-MS) can also identify previously known protein–protein interactions as well as suggest potential interacting partners that have not been previously described.

If a binding partner detected by co-IP truly interacts with a particular target protein, then multiple primary antibodies specific for the same epitope on that target protein should yield the same results. Antibodies that bind the same target protein but differ in epitope specificity may also co-IP the same proteins, although antibodies are known to prevent or disrupt the protein–protein interactions of protein complexes. Another indicator of a true protein–protein interaction, as opposed to an artifact, is that either protein can be co-immunoprecipitated when the IP antibody against the binding partner is used (i.e., protein A can be used to co-IP protein B, and protein B can be use to co-IP protein A).

Even high quality, monoclonal antibodies may bind to nonspecific proteins; therefore, performing a co-IP using a non-target antibody (often referred to as an 'irrelevant antibody') is critical to confirm that the immunoprecipitated protein complex is the specific complex that was sought. And because antibody specificity varies by subclass, it is recommended to use control antibodies that match the primary antibody as close as possible. 

Many protein–protein interactions are dependent upon the activation of one or more of the binding partners in a complex. Therefore, to test if a true interaction occurs, cells that express an inactive variant of one of the binding partners can be used for co-IP of the protein complex; if activation is required, then the complex will not be co-precipitated with the target antigen.

Cell lysis causes proteins that never interact to come into close association, and it is inevitable that some proteins will bind to each other. To test if a detected protein complex forms after cell lysis, Ohh et al. metabolically labeled all proteins in cells and then lysed the cells with a lysis buffer that included the purified, unlabeled form of the protein of interest. Because this unlabeled protein was unable to compete with the radiolabeled protein for complex formation recovered by co-IP, the researchers concluded that the complex represented a physiologically relevant interaction that formed prior to lysis.