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IP vs. Co-IP

Immunoprecipitation

The topic of co-immunoprecipitation (Co-IP) is best preceded by a discussion of immunoprecipitation (IP) to help frame an understanding of the principles involved. Immunoprecipitation is one of the most widely used methods for antigen detection and purification. An important characteristic of IP reactions is their potential to deliver not only the target protein but also other macromolecules that interact with the target.

The principle of an IP is very simple. An antibody (monoclonal or polyclonal) against a specific target antigen is allowed to form an immune complex with that target in a sample, such as a cell lysate. The immune complex is then captured on a solid support to which either Protein A or Protein G has been immobilized (Protein A or G binds to the antibody, which is bound to its antigen). The process of capturing this complex from the solution is referred to as precipitation. Any proteins not “precipitated” by the immobilized Protein A or G support are washed away. Finally, components of the bound immune complex (both antigen and antibody) are eluted from the support and analyzed by SDS-PAGE (gel electrophoresis), often followed by Western blot detection to verify the identity of the antigen.

Traditional immunoprecipitation involves the following steps:

1. Form the antigen-antibody complex (immune complex) by incubating specific antibody with the antigen-containing sample for 1 hour to several hours.
2. Capture the immune complex on an immobilized Protein A or Protein G agarose gel support by incubation for 0.5-2 hours.
3. Remove any non-bound protein (non-immune complex sample components) from the precipitated complex by washing gel support with additional sample buffer.
4. Boil gel support in reducing SDS-PAGE sample loading buffer.
5. Recover sample eluted in loading buffer from gel support and analyze by SDS-PAGE.
6. Perform Western blot analysis, probing with antigen-specific antibody

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Overview of affinity purification

Immunoprecipitation Kit
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Tech Tip #64: Immunoprecipitation Technical Guide and Protocols

Summary of a traditional immunoprecipitation procedure.

Co-Immnunoprecipitation

Co-immunoprecipitation (Co-IP) is a popular technique for protein interaction discovery. Co-IP is conducted in essentially the same manner as an IP. However, in a co-IP the target antigen precipitated by the antibody “co-precipitates” a binding partner/protein complex from a lysate, i.e., the interacting protein is bound to the target antigen, which becomes bound by the antibody that becomes captured on the Protein A or G beads. The assumption that is usually made when associated proteins are co-precipitated is that these proteins are related to the function of the target antigen at the cellular level. This is only an assumption, however, that is subject to further verification.

Summary of a co- immunoprecipitation (Co-IP) procedure.

Traditional Methods vs. Innovations for Co-IP

Problems with Traditional Co-IP Methods

The traditional co-IP protocol has certain deficiencies relating to the fundamental format of the assay, the antibody and associated chemistry. One of the most commonly encountered problems with the traditional IP and co-IP approach is interference from antibody bands in gel analysis. In those cases where several proteins may be co-precipitated with the target, presence of the co-eluted antibody heavy and light chains (25 and 50 kDa bands in reducing SDS-PAGE gel) in the preparation can obscure the results. The ideal situation would be to conduct the co-IP without contamination of the eluted antigen with antibody. With this potential interference eliminated, only the co-precipitated proteins will be present and detected on a gel.

Approaches to Co-IP Free of Antibody Interference

Antibody Orientation and Immobilization – Indirect Strategy

This strategy takes advantage of the binding characteristics of the traditional Immobilized Protein A or Protein G based support combined with chemical cross-linking to covalently link the antibody to the support. Protein A and Protein G bind IgG class antibodies through the Fc region which is characterized primarily by dimerized heavy chain modified by carbohydrate. Fc region binding naturally orients the antigen binding domains of the antibody (Fab) away from the support, making them available for binding to their respective target antigen. To ensure that the antibody remains on the support during the requisite antigen binding, wash and elution steps of the protocol, this bound and oriented antibody is chemically crosslinked to the Protein A or G with the bifunctional reagent, disuccinimidyl suberate (DSS).

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Immunoprecipitation Kit
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Crosslink IP Kit

Activated Support for Antibody Immobilization – Direct Strategy

In this approach, antibody is immobilized directly through its surface amine groups (contributed primarily by the side chain e -amino group of lysine) to a high capacity aldehyde activated beaded agarose support (AminoLink Plus Coupling Resin). The support forms a Schiff’s base with these available amines that is reduced to form stable secondary amine bonds during the immobilization process. The wide range of coupling conditions that can be used with this support make it ideal for maintaining biological binding activity critical to the successful execution of a co-IP experiment.

HA and c-Myc Tag Targeted-Direct Strategy

Given the immense number and variety of different protein targets that researchers may wish to immunoprecipitate, most researchers have to immobilize their own primary antibodies using one of the indirector direct strategies discussed here. 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 available. Specific antibodies against the HA tag (YPYDVPDYA) or c-Myc tag (EQKLISEEDL) can be covalently immobilized to beaded agarose resin, enabling use in IP or co-IP experiments involving HA- or c-Myc-tagged “bait” proteins.

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HA Tag IP/Co-IP Kit

c-Myc Tag IP/Co-IP Kit

Co-IP of active Rac1 with HA-tagged Pak1-PBD (p21 binding domain). Human 293 cells were transfected with HA-Pak1-PBD alone or co-transfected with constitutively activated Rac1 (Q61L). Anti-HA agarose slurry (6 µl) was incubated with 50 µl HA-tagged positive control lysate from the kit (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 (panel A) and then reprobed with anti-HA antibody (panel B).

Streptavidin-Biotin Approaches

This direct coupling approach incorporates the binding association between streptavidin and biotin. Streptavidin immobilized to beaded agarose resin or coated in microplate wells provides an alternative IP or co-IP strategy for obtaining results free from antibody interference. Biotinylated antibody is bound very strongly to each matrix and is not eluted when mild conditions are used to release the target antigen. The IP/co-IP is conducted by incubating the sample with the streptavidin-biotinylated antibody loaded matrix. Elution of the target antigen and any interacting proteins is performed free of antibody contamination. A wide selection of affinity resins, magnetic beads and coated plates based on immobilized avidin, streptavidin or Thermo Scientific NeutrAvidin Proteins facilitate this strategy.

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Avidin and Streptavidin
Agarose Resins

Biotinylated Protein
Interaction Pull-Down Kit

Optimization and Evaluation for IP and Co-IP

Classical Immune Complex Formation vs. Pre-binding of Antibody

A change in protocol from the classical immune complex precipitation is necessary when using immobilized antibody in the co-IP method. In the traditional co-IP protocol, the immune complex (antigen:antibody) is formed in solution before “precipitating” it with the immobilized Protein A or Protein G matrix. With the antibody pre-attached to the “precipitating” matrix, the immune complex is formed directly on the matrix by incubation of the antigen-containing sample with the matrix. Formation of the immune complex (the target antigen and any target-associated protein) and its precipitation occurs in one step.

Our in-house application research suggests that, even for traditional (classic) immunoprecipitation procedures that do not require it, pre-binding the antibody consistently results in the capture of more target antigen. In the immobilized format, the antibody is allowed to incubate with the lysate for the specified period. With Crosslink and Direct IP Kits, the matrix is washed using a spin cup format and the bound protein eluted in a separate step prior to analysis. The target antigen and co-IP complex is recovered free of antibody or antibody fragment contamination, and the antibody is retained in an active form on the support to be used in another co-IP cycle.

Evaluating a Co-IP Captured Interaction

In their review of protein interactions, Phizicky and Fields present a discussion of the issues to consider in validating a suspected interaction obtained by a co- IP experiment. Ultimately, the following question must be answered: Does the interaction detected by co-IP occur in vivo , and what significance does it have at the cellular level? A summary of Phizicky and Fields approach to verification of co-IP data follows.

Confirm that the co-precipitated protein is obtained only by antibody against the target. Use monoclonal antibodies in the co-IP protocol. When only a polyclonal antibody is available, pre-treatment of the antibody with sample devoid of the primary target (bait protein) may be required to assure that the polyclonal antibody does not contain clones or contaminants that bind prey protein(s) directly. Pre-adsorption to extracts devoid of target or pre-purification of polyclonal IP antibodies against an affinity column containing pure target antigen safeguards against a false positive co-IP.

Verify that antibody against the target antigen does not itself recognize the co-precipitated protein(s). Use independently derived antibodies that have demonstrated specificities against different epitopes on the target protein. Their use serves as verification that the target (bait)-directed antibodies have no affinity for the target associated prey proteins recovered during the co-IP. Alternatively, an antibody against the co-precipitated protein can be used to co-IP the same complex.

Determine if the interaction is direct or indirect. Is the interaction mediated through a third party protein that contacts both target and co-precipitated protein? Immunological and other more sophisticated methods such as mass spectrometry may be necessary to answer this question.

Determine that the interaction takes place in the cell and not as a consequence of cell lysis. Suggested approaches here involve co-localization studies and site-specific mutagenesis giving rise to mutants that perturb the binding process.

References

1. Liebler, D.C. (2002). Identifying protein-protein interactions and protein complexes. In Introduction to Proteomics, Tools for the New Biology, Humana Press, pp.151- 165. (Product # 20061)
2. Adams, P.D., et al. (2002). Identification of Associated Proteins by Coimmunoprecipitation, In Protein-Protein Interactions – A Molecular Cloning Manual. Golemis, E., Ed., Cold Spring Harbor Laboratory Press, pp 59-74. (Product # 20068)
3. Phizicky, E.M. and Fields, S. (1995). Protein-Protein Interactions: Methods for Detection and Analysis. Microbiological Reviews (Mar.), pp. 94-123.