Immunoprecipitation (IP) vs. Co-immunoprecipitation (Co-IP)
The topic of co-immunoprecipitation (Co-IP) is best preceded by a review 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.
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).
Co-IP Optimization Strategies
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 Protein Methods Library. Buffers with low ionic strength (i.e., <120mM NaCl) that contain non-ionic detergents (NP-40 and Triton X-100) are less likely to disrupt protein-protein interactions; however, empirical testing may be required to determine the best buffer formulation for a specific protein complex of interest.
Additionally, lysing cells by sonication or vortexing the lysate or bead-bound immune complexes during the wash steps should be avoided to prevent the disruption of the protein-protein interaction(s) of the target complex. And while centrifugation is a standard method to separate the precipitated complexes from the remaining lysate and during wash steps, the samples should be handled gently to prevent the loss of bound complex proteins.
An advanced technique to strengthen protein-protein interactions is by crosslinking the binding partners. Using this approach, all proteins within the active distance of the specific reagent in a cell lysate are covalently crosslinked, and the target protein can then be immunoprecipitated along with the other proteins in the complex without the risk of losing binding partners.
With the myriad of proteins in cell lysates, it is inevitable that nonspecific binding to the IP antibody will occur, especially when using the batch method (a gentle, large-scale procedure) of immunoprecipitating the target protein. Additionally, because proteins that are normally separated into discrete cellular compartments are now mixed together, nonphysiological binding to the target complex is likely to occur, especially with abundant proteins such as actin. These nonspecific interactions are often broken by thoroughly washing the bead-bound immune complexes, but other strategies may be applied to optimize nonspecific binding, including:
- Changing the ionic strength of the IP buffer by titrating the salt concentration from 120 to 1000mM.
- Decreasing the amount of primary antibody until the signal:noise ratio is maximized.
- Preclearing the lysates, as described in the Immunoprecipitation section.
- Tech Tip #4: Batch and spin cup methods for affinity purification of proteins
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 50kDa 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 Immunoprecipitation 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.
|Traditional Co-IP Problems||Solutions|
|Batch processing of the precipitated complex in a single tube: results in inefficient washing of non-bound proteins from the support and resin loss due to decanting wash buffer from the tube via a pipette.||Spin cup or spin tube processing: dedicated IP and co-IP kits that contain spin-cup or spin-tube devices that increase washing efficiency, offer more effective elution of antigen and associated proteins and eliminate resin loss yielding more consistent results.|
|Antibody fragment interference: co-elution of antibody fragments with antigen often results in bands interfering with the detection of any co-precipitated proteins by SDS-PAGE.||Antibody immobilization: chemistries designed to immobilize the antibody to the support, thereby allowing the elution of only the target and any associated proteins in a co-IP complex.|
|Antibody sacrificed: as a consequence of harsh elution conditions, the target antibody is destroyed; antibody loss by way of the protocol can be costly.||Antibody re-used: immobilization chemistry and mild elution conditions for the target and associated proteins allow the immobilized antibody to be re-equilibrated and re-cycled several times in the co-IP protocol.|
Evaluating a Co-immunoprecipitated Protein-Protein Interaction
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 (1,2).
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 noncharacterized 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.
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.
Protein Interactions Technical Handbook
Our 72-page Protein Interactions Technical Handbook provides protocols and technical and product information to help maximize results for protein interaction studies. The handbook provides background, helpful hints and troubleshooting advice for immunoprecipitation and co-immunoprecipitation assays, pull-down assays, far-western blotting and crosslinking. The handbook also features an expanded section on methods to study protein–nucleic acid interactions, including ChIP, EMSA and RNA EMSA. The handbook is an essential resource for any laboratory studying protein interactions.
Contents include: Introduction to protein interactions, Co-immunoprecipitation assays, Pull-down assays, Far-western blotting, Protein interaction mapping, Yeast two-hybrid reporter assays, Electrophoretic mobility shift assays (EMSA), Chromatin immunoprecipitation assays (ChIP), Protein–nucleic acid conjugates, and more.
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 (3).
- Phizicky E. M. and Fields S. (1995) Protein-protein interactions: Methods for detection and analysis. Microbiol Rev. 59, 94-123.
- Golemis E. (2002) Protein-protein interactions : A molecular cloning manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. ix, 682.
- Ohh M. et al. (1998) The von hippel-lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol Cell. 1, 959-68.
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