Co-Immunoprecipitation | Antibodies Inc.

Co-Immunoprecipitation is an extension of the basic IP technique [1] which has been developed to identify and study biologically relevant protein-protein interactions [2-7]. The basic principle underlying co-immunoprecipitation (Co-IP) is the same as for immunoprecipitation (IP); a target protein is captured using a specific antibody which is then precipitated using beads. In basic IP, the primary interest is in the captured target protein. However, in Co-IP, the primary interest is in identifying and studying the biologically relevant protein(s) that bind to the target protein and are co-precipitated with it.

The basic IP protocol involves the following steps:

Sample preparation: A cell or tissue lysate is prepared. This a homogenized mixture of cells containing all cellular components, including the protein of interest (the target protein). Normally, the lysate would be prepared under conditions that retain the target protein in its native, folded state. Note: This is crucial for Co-IP to ensure that protein-protein interactions are maintained, and protein complexes remain intact.
Antibody binding: The lysate is incubated with an antibody that binds tightly to an epitope on the surface of the native target protein. This results in the formation of an antibody-target protein complex.
Immunoprecipitation: The antibody-target protein complex is then captured, ready for subsequent analysis. The complex is captured by immobilizing the antibody on a solid support, often Protein A or Protein G Sepharose beads. The antibody can be bound to the beads either prior to incubation with the lysate, or at the end of the incubation period. The beads are typically captured by centrifugation (though it is also possible to use magnetic beads). It is also possible to use specifically designed reagents for capturing the immune complex. For example, our PrecipHen® Immunoprecipitation Reagent (Catalog P-1010) uses Goat Anti-Chicken IgY antibodies covalently attached to agarose beads to precipitate chicken antibodies used in IP experiments.
After several washing steps to remove non-specifically bound proteins, the captured antibody-target protein complex is released from the beads using a suitable elution buffer.
Analysis: The eluted proteins are typically analyzed by Western Blot in a standard IP experiment.

As discussed above, in Co-IP, the primary interest is in the biologically relevant proteins and proteins complexes that are bound to and co-precipitate with the target protein.

Typical applications of Co-IP include:

Identification and characterization of protein-protein interactions, providing insights into the structural and functional organization of cellular pathways.
Investigation of the role of post-translational modifications and their impact on protein interactions. By isolating protein complexes under different cellular conditions or in response to stimuli, the role of modifications such as phosphorylation, acetylation, or ubiquitination in modulating protein interactions and cellular signaling may be explored.
Exploring the role of the dysregulation of protein-protein interactions in disease processes.

Key experimental considerations

Just as with the basic IP technique, Co-IP relies on the specificity of antibodies to recognize and bind to their target proteins. Antibodies are chosen based on their ability to selectively bind to a particular protein of interest within the complex mixture of cellular proteins in a typical cell lysate. It is crucial that the antibody used in a Co-IP experiment is highly specific for the target protein and that it does not bind to non-target proteins.

Both monoclonal and polyclonal antibodies can be used, each having advantages and disadvantages. Polyclonals will usually contain antibodies against multiple epitopes which can lead to tighter and more rapid binding of the target protein. Monoclonals will recognize a single epitope. This can be advantageous, as binding to certain epitopes may disrupt protein-protein interactions. This is more easily controlled for with an antibody recognizing a single epitope. If possible, select an antibody that has already been validated in IP (to avoid having to validate it prior to performing your Co-IP experiment). Examples from our catalog are our mouse monoclonal Anti-PSD-95 Antibody (K28/43) (Catalog 75-028) and our rabbit polyclonal Anti-PKCα (Ser-657/Tyr-658), Phosphospecific Antibody (Catalog PP1091).

Additionally, Co-IP depends crucially on the maintenance of biologically relevant protein-protein interactions, both when the cells are lysed and throughout the subsequent isolation steps. The stronger the protein-protein interaction, the more likely it is to survive extraction and isolation. Low affinity and transient protein-protein interactions may not survive unless they can be stabilized (see Hints and Tips, below).

There are two aspects to preserving protein-protein interactions. First, protein-protein interactions may be disrupted during cell lysis and subsequent processing due to the composition of lysis / wash buffers, or the use of harsh mechanical methods of cell disruption. Mild cell lysis conditions must be employed. Many protein-protein interactions will be stable in lysis buffers of physiological pH containing low levels of gentle non-ionic detergents such as NP-40, Tween 20 or Triton X-100 and physiological or lower concentrations of NaCl. Whilst this is a good starting point, extensive buffer optimization may be required. Harsh detergents such as SDS and sodium deoxycholate should be avoided as they will tend to disrupt protein-protein interactions. For soluble proteins it may be possible to omit detergents from the lysis buffer. This may help preserve weaker protein-protein interactions, though either freeze-thaw or some form of gentle mechanical disruption may be required to lyse cells in the absence of detergent. A general rule of thumb is to treat the lysate as gently as possible; sonication or vortexing are likely to disrupt weak protein-protein interactions and should be avoided.

Second, the protein-protein interaction of interest may be dependent on post-translational modifications (PTMs) of the proteins involved. Therefore, it is important to maintain PTMs throughout the Co-IP experiment. Lysis and elution buffers should contain suitable inhibitors to block, for example, dephosphorylation, deubiquitylation, deacetylation or the removal/addition of any other PTM that may be relevant to the experiment. Additionally, it is advisable to include a broad-spectrum proteinase inhibitor cocktail to prevent protein degradation.

Whilst extensive washing of the beads is essential to remove proteins bound non-specifically, gentle conditions must be used to preserve the physiologically relevant interactions of interest.

The protein complexes must be eluted from the beads prior to analysis, typically either via Western Blot or mass spectrometry.

Analysis

The CoIP experiments are often analyzed by Western Blot. As with standard IP, if the Western Blot primary antibody is from the same species as the IP antibody, the Western Blot secondary antibody will detect the heavy (~55kDa) and light (~25 kDa) chains of the IP antibody (the IP antibody is usually eluted from the beads along with the captured proteins of interest) . This will pose a problem if the proteins of interest are close to these sizes. This can be avoided by using a Western Blot primary antibody from a different species to the IP antibody.

A consideration unique to Co-IP is the question of what antibody to use to probe the Western Blot – i.e. what proteins should be probed for? If investigating a known protein interaction, then it will be straightforward to choose an appropriate Western Blot antibody. However, when investigating a novel interaction, the identity of the protein / protein complex binding to the target protein may be unknown.

Co-IP experiments may also be analyzed by mass spectrometry, which has the advantage of being able to identify components of a protein complex without the need for any prior knowledge as to their likely identity.

Validation of results

Whilst Co-IP is a powerful technique for identifying protein-protein interactions, care must be taken to validate the results and establish that the interactions identified are biologically relevant and not experimental artifacts.

As a first step, negative controls should be run to correct for non-specific binding of proteins.

Key negative controls would be:

Protein A/G beads only – to identify proteins binding non-specifically to the beads used for the pull-down.
IP performed with an isotype control – to control for non-specific interactions with the class of antibody used.

Additional validation experiments should be run if possible. Ideally, an orthogonal approach should be taken to validating the Co-IP results. If the epitope recognized by the capture antibody is known, other antibodies recognizing the same epitope should produce similar results. Additionally, if pull-down of protein X identifies protein Y as a binding partner, then pull-down of protein Y (if a suitable antibody exists) should identify protein X as a binding partner.

Consideration should also be given as to whether an identified protein-protein interaction makes biological sense in the intact cell. Once a cell is lysed, and the normal subcellular compartmentalization is disrupted, proteins that would never be in physical contact in a live cell may now interact in the homogenized cell lysate, giving rise to non-biologically relevant interactions.

Enhancements of the Co-IP technique

One issue with the use of Protein A/G beads to pull down the capture antibody, is that methods used to release the target protein (and associated proteins) from the capture antibody will also release the capture antibody from the Protein A/G beads; as discussed above, the presence of the capture antibody may be problematic during subsequent analysis. One way to overcome this is to chemically immobilize the capture antibody directly onto beads. This way, so long as non-reducing elution conditions are used, no antibody / antibody fragments will be released into the eluate along with the isolated protein complex. However, immobilization of the antibody in this way may reduce the efficiency of target protein capture.

In a variation of the Co-IP technique, the protein of interest is recombinantly expressed with a tag (for example, HA, c-Myc, FLAG or GFP)[3 - 5]. The immunoprecipitation step is then performed using the appropriate anti-Tag antibody. This has the advantage that there are many anti-Tag antibodies available with high affinity and high specificity. This approach can be particularly useful for targets for which good quality IP antibodies do not exist. A potential downside to this approach is that one is no longer looking at protein-protein interactions involving the endogenous target protein, thus the biological relevance of interactions identified in this way could be called into question.

Typical Co-Immunoprecipitation Workflow

Lysate preparation

Cells: Lyse cells by addition of non-denaturing lysis buffer (typically 20 -50 mM Tris HCl, pH 7.5- 8.0; 150 mM NaCl; 0.5-1.0% NP-40; 2 mM EDTA; broad-spectrum proteinase inhibitors cocktail; protein phosphatase inhibitors; other inhibitors to maintain relevant PTMs). Incubate at 4°C for 30 - 120 minutes with constant gentle agitation.
Pellet cellular debris and retain supernatant for Co-IP experiment.
Tissue: Tissue should be rinsed with PBS to remove blood, finely chopped with scissors, and then homogenized in lysis buffer. Ideally, gentle disruption of the tissue using a Dounce homogenizer is preferable to harsher methods such as sonication or electric tissue homogenizers. Incubate on ice for 30- 120 minutes with constant gentle agitation. Pellet cellular debris and retain supernatant for Co-IP experiment.

Antibody incubation

Method 1: (incubate with free antibody): Add the antibody against the target protein at the manufacturer’s recommended dilution and incubate on ice with gentle agitation at 4°C for 1 hour - overnight. Prepare Protein A / G beads by washing in lysis buffer. Add the washed beads to the lysate and incubate for 1-4 hours at 4°C with gentle agitation. Pellet the beads by centrifugation to capture the protein-antibody immune complex.
Method 2: (incubate with immobilized antibody): Incubate the required amount of antibody with Protein A / G beads at 4°C for 1-4 hours with gentle agitation to allow binding of the antibody to the Protein A / G. Wash with lysis buffer. Add the washed beads to the lysate and incubate as for Method 1.
Method 3: If using an immobilized secondary antibody (such as our PrecipHen® Immunoprecipitation Reagent (Catalog P-1010)), either Method 1 or Method 2 can be followed.
Note: Capture of the target protein and interacting proteins may be more efficient if the antibody is incubated with the lysate prior to immobilization on Protein A / G beads or secondary antibody beads.

Washing

The pelleted antibody-beads have hopefully captured the target protein and associated biologically relevant protein complexes. The beads now need to be washed (typically 3 times) to remove unbound / contaminating proteins.

Elution

To analyze the proteins bound to the beads, they must first be eluted. Three different elution conditions are frequently used, depending on the requirement of the experiment.

SDS-PAGE sample buffer
A denaturing buffer that efficiently elutes target protein(s) and antibody from Protein A/G beads. Simple and efficient for analyzing samples by SDS-PAGE/Western Blot
Low pH glycine (0.1 M glycine, pH 2.5 – 3.0)
This buffer breaks most antibody-target protein interactions and antibody-Protein A/G interactions. The eluted proteins should be immediately neutralized to avoid denaturation. Eluted proteins may retain functional (e.g. enzymatic activity) which provides a potential alternative method of analysis.
Urea (6-8 M urea, 20-50 mM Tris-HCl pH 7.5, 100 mM NaCl)
This method can be useful if samples are to be analyzed by mass spectrometry as it is compatible with proteinase digestion protocols.

If Co-IP is performed using a tagged protein, such as FLAG or HA, then the tagged protein can be eluted from the immobilized capture antibody gently and specifically using FLAG or HA peptide.

Regardless of which elution method is used, the beads are removed by centrifugation and the supernatant taken forward for analysis.

Analysis

The eluted proteins are typically analyzed by SDS-PAGE/Western blot or mass spectrometry.

Hints and Tips

How to reduce non-specific background

One way to reduce non-specific protein binding and reduce background in IP and Co-IP experiments is to pre-clear the lysate prior to incubating the lysate with the capture antibody. In this procedure, the lysate is incubated with an off-target isotype control antibody and Protein A/G beads (or whatever type of beads will be used in the actual CoIP experiment). The isotype control-beads complex is removed by centrifugation, and the supernatant used in the Co-IP experiment.

It is also possible to reduce non-specific protein binding by pre-blocking the beads to be used in the Co-IP experiment. In the same way that the membrane in a Western Blot experiment is blocked prior to use, the beads can be pre-incubated with 1-3% BSA for 1-2 hours at 4°C (then washed to remove free BSA) prior to addition to the Co-IP lysate.

How to stabilize and identify weak and transient protein complexes

Weak and transient protein-protein interactions can be difficult to identify by Co-IP due to their instability and loss during the processing of the samples. One approach is to use cross-linking reagents to stabilize/freeze the protein-protein interaction(s) prior to cell lysis. Wang et al used dithiobis succinimidyl propionate (DSP), a cell membrane permeable cross-linker, which has an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of a cleavable spacer to cross-link co-localized proteins in intact cells prior to lysis and IP [6]. It is also possible to cross-link and stabilize protein complexes after cell lysis [7]

Handy hint

When washing the beads at the end of the antibody incubation step, retain all the washes. It may be informative to analyse these if the target protein/ known binding partners are not precipitated as expected.

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Guide to Co-Immunoprecipitation

Principles of Co-Immunoprecipitation

Typical Co-Immunoprecipitation Workflow

Hints and Tips

Further Reading