Immunoprecipitation and co-immunoprecipitation protocol

Immunoprecipitation (IP) is a protein purification method that allows you to isolate a specific protein from a mixture of proteins using antigen-antibody interactions, where the specific protein being isolated is often referred to as the target antigen. This technique is also considered a form of affinity purification. Immunoprecipitation is widely used to isolate proteins from complex mixtures.

An antibody for the protein of interest is incubated with a cell extract, enabling the antibody to bind to the protein in solution. In co-immunoprecipitation (co-IP) experiments, the protein of interest is often called the bait protein. The antibody/antigen complex is then pulled out of the sample using protein A/G-coupled agarose or magnetic beads. This isolates the protein of interest from the rest of the sample. The protein of interest can then be analyzed by western blot, mass spectrometry, direct ELISA, or other analytical techniques. IP and co-IP are related methodologies commonly used to study protein-protein interactions.

Introduction to immunoprecipitation

Immunoprecipitation (IP) is a cornerstone technique in molecular biology for isolating and studying specific proteins from complex biological samples. By leveraging the highly specific interaction between an antibody and its target protein, researchers can selectively capture the protein of interest from a mixture, enabling detailed analysis of protein complexes and protein-protein interactions. This approach is invaluable for identifying interacting proteins, mapping protein interaction networks, and dissecting the composition of protein complexes within cells. The success of immunoprecipitation relies on careful selection of antibodies that are specific to the target protein, as well as the optimization of lysis buffers to preserve native protein interactions. Downstream analyses, such as western blotting and mass spectrometry, are commonly used to characterize the isolated proteins and their binding partners, providing insights into cellular signaling pathways, regulatory mechanisms, and disease processes. As a result, immunoprecipitation remains an essential tool for advancing our understanding of protein function and interaction in biological systems.

Stage 1 - Preparing the lysates

The preparation of cell or tissue lysates is a foundational step in immunoprecipitation, as it directly impacts the ability to isolate intact protein complexes and study protein interactions. Effective cell lysis requires the use of carefully formulated lysis buffers that solubilize proteins while preserving their native structure and interactions with other proteins. Non-ionic detergents, such as NP-40 and Triton X-100, are commonly included in lysis buffers to gently disrupt cell membranes and release soluble proteins without denaturing them. The salt concentration, pH, and inclusion of protease and phosphatase inhibitors are also critical for maintaining protein stability and preventing degradation or dephosphorylation during extraction. In some cases, ionic detergents may be used for more stringent lysis, but these can disrupt protein-protein interactions and should be chosen based on the specific requirements of the experiment. Optimizing the lysis buffer for your sample type and target protein ensures that protein complexes and interacting proteins remain intact, setting the stage for successful immunoprecipitation and downstream analysis.

The first step is to lyse the cell or tissue samples in a suitable buffer to release proteins into the solution. We provide a non-denaturing lysis buffer (ab152163) suitable for isolating most proteins from cells or tissues.

Some proteins are more difficult to isolate than others, so you may need to create and optimize your own buffer. The ideal lysis buffer will minimize protein denaturation while releasing enough proteins from the sample.

Non-ionic detergents, such as NP-40 and Triton X-100, are less harsh than ionic detergents, such as SDS and sodium deoxycholate. Other variables that can affect the success of immunoprecipitation include salt concentration, divalent cation concentration, pH, and temperature.

Materials required

• Suitable lysis buffer (example: ab152163, or develop your own)
• Protease inhibitor cocktail (example: ab65621)
• Phosphatase inhibitor cocktail (optional – example: ab201112)
• PBS

Steps

Prepare an appropriate lysis buffer for your protein.

Add protease inhibitors to the buffer.

• Include phosphatase inhibitors for phosphorylated proteins.

Isolate your cells and suspend them in a lysis buffer.

• You should prepare a suspension with 300 µL of the lysis buffer added for 1–3 x10⁷ cells or 600 µL of the buffer added for more than 3 x10⁷ cells.

  • Some adherent cells may require enzymatic or mechanical detachment. We detach adherent cells with TrypLE, spin to remove the media, resuspend the pellet in PBS and spin two more times, each time resuspending the pellet in PBS to wash the cells, leaving a washed cell pellet at the end.
  • Suspension cells can be washed in PBS and spun down into a pellet before suspension in the ice-cold lysis buffer in the next step.

To lyse the cells, add ice-cold lysis buffer directly to the cell pellet and resuspend it.

• Incubate the cells with lysis buffer on ice for 10 mins (without agitation).
• Sonicate the lysate 3 times in ice-cold water.

Spin down the suspension to pellet insoluble contents.

• Keep the supernatant – this is your lysate.
• Centrifuge the suspension at 8,000 x g for 10 minutes at 4°C.
• Place the supernatant in a fresh tube on ice.

Determine the protein concentration in your lysate using a Bradford or BCA assay.

• Suppose the protein concentration at this stage is low, and your protein resides in the nucleus or mitochondria. In that case, you could consider fractionating your original sample to produce a more concentrated lysate.
• We offer cell fractionation kits for this purpose.

If not using immediately, snap freeze aliquots in liquid nitrogen and store at -80°C.

Materials required

• Suitable lysis buffer (example: ab152163, or develop your own)
• Protease inhibitor cocktail (example: ab65621)
• Phosphatase inhibitor cocktail (optional – example: ab201112)
• PBS
• Electric homogenizer

Steps

Prepare an appropriate lysis buffer for your protein.

Add protease inhibitors to the buffer.

• Include phosphatase inhibitors for phosphorylated proteins.

Snap freeze the tissue and break it up with the bead beater homogenizer before lysis.

• Smash frozen tissues into powder using BioPulverizer on dry ice and add 200 mg of the tissue to the bead beater tube.

Lyse the cells in the bead beater.

• Add 1200 µL of lysis buffer to the bead beater tube containing the 200 mg of the tissue sample.
• Insert the tubes in the bead beater and homogenize for 1 min 30 secs.
• Remove the tubes and place them on ice for 1 minute to ensure they are cold.
• Add the tubes to the bead beater for a second homogenization step for a further 1 min 30 secs.
• After this, incubate on wet ice for 5 mins before spinning.

Spin down the suspension to pellet insoluble contents. Keep the supernatant – this is your lysate.

• Centrifuge the suspension at 8,000 x g for 10 minutes at 4°C.
• Place the supernatant in a fresh tube on ice.

Determine the protein concentration in your lysate using a Bradford or BCA assay.

• Suppose the protein concentration at this stage is low, and your protein resides in the nucleus or mitochondria. In that case, you could consider fractionating your original sample to produce a more concentrated lysate.
• We offer cell fractionation kits for this purpose.

If not using immediately, snap freeze aliquots in liquid nitrogen and store at -80°C.

Stage 2 - Pre-clearing the lysates (optional)

Pre-clearing lysates is an additional optional step that can help to increase the purity of proteins obtained by IP. This step involves incubating the lysate with only beads or beads plus an isotype control, depending on the beads used, to precipitate unwanted proteins. When choosing a control antibody, it is important to use an antibody from the same species as the experimental antibody to serve as an appropriate negative control. Including a negative control, such as normal IgG, is essential to distinguish specific from non-specific binding in co-IPs. One can normally skip this step when using high-quality beads without a severe bead adsorption effect.

Pre-clearing the lysate can help reduce non-specific binding and background. However, if the final detection of the protein is by western blotting, pre-clearing may not be necessary unless a contaminating protein interferes with the visualization of the protein of interest. In co-IPs, multiple controls, including isotype and negative controls, are often required to validate the specificity and reliability of the results.

Here we provide a protocol example for pre-clearing the lysates using beads combined with an isotype control.

Materials required

• Isotype control antibody
• Your lysate
• Protein A/G magnetic beads (example ab214286)
• Magnetic rack

Steps

Prepare the beads and mix them with isotype control antibodies.

• Follow the bead manufacturer’s advice to prepare the antibody-bead complexes; in particular, the optimal binding ratio of antibody and beads.

Add a slurry of beads with isotype control antibodies to the lysate.

• Add ~100 µL of beads per 1 mL of lysate.
• Incubate for 10 – 30 mins at 4°C with gentle agitation.

Remove the antibody-bead complexes.

• Place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
• Aspirate the solution, leaving the beads behind, and transfer this solution to a new tube.

Proceed to IP on your pre-cleared lysate.

Materials required

• Isotype control antibody
• Your lysate
• Protein A/G beads (example ab193256)
• Microcentrifuge

Steps

Incubate the isotype control antibody with lysate overnight.

Add the slurry of agarose beads to the antibody-lysate mix and incubate for another 2 hours.

• Add ~100 µL of beads per 1 mL of lysate.
• Incubate for 2 hours at 4°C, with gentle agitation.

Remove the antibody-bead complexes by light centrifugation.

• Spin down the tube at the manufacturer's recommended speed for 2 minutes at 4°C.
• Aspirate the solution, leaving the beads behind, and transfer this solution to a new tube.

Proceed to IP on your pre-cleared lysate.

Stage 3 - Immunoprecipitation and washing

Now we have lysed your cells and pre-cleared the lysate; if necessary, we are ready to run the IP.

There are two main methods to immunoprecipitate proteins. The first approach is mixing the antibody with the lysate and then adding Protein A/G beads to the antibody-lysate complex. This method yields high purity of protein; however, the co-IP antibody or IP antibody can also be co-eluted with the protein of interest, which sometimes creates difficulties in western blot detection due to antibody contamination in downstream analysis.

The second approach involves preparing the antibody-bead complexes and incubating them with the lysate to extract the protein of interest. This method yields less than the first one but avoids the problem of co-elution of antibodies, reducing antibody contamination.

When selecting an antibody, it is important to choose an antibody specific to your target protein. Using a polyclonal antibody can offer advantages, such as recognizing multiple epitopes, which may improve capture efficiency and increase the likelihood of successful co-IP. Consider the binding affinities and binding sites between the antibody and antigen, as these factors influence the effectiveness of the interaction.

We suggest you run IP according to the protocols below. If using an isotype control antibody, you can run the same procedure and compare this with your IP for the protein of interest when analyzing the results. Optimizing these steps is crucial for achieving successful co-IP and successful co results.

Materials required

• Your lysate
• Primary antibody
• Isotype control antibody
• Protein A/G magnetic beads (example: ab214286)
• Magnetic rack

Steps

Incubate your lysate with antibodies at 4°C overnight, with gentle agitation.

Add the Protein A/G magnetic beads to the antibody-lysate complex.

• Add ~100 µL of beads per 1 mL of lysate.
• Incubate for 1–4 hours at 4°C, with gentle shaking.

Wash the beads three times with PBS or another wash buffer of your choice to remove unbound impurities.

• Place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
• Aspirate and discard the solution, keeping the beads in the tube.
• Add ~ 1 mL of fresh lysis buffer to the beads and repeat the steps above.

Materials required

• Your lysate
• Primary antibody
• Isotype control antibody
• Protein A/G beads (example: ab193256)
• Microcentrifuge

Steps

Prepare the antibody-lysate complex.

• Incubate the lysate with antibodies at 4°C overnight, with gentle swinging.

Add the beads to antibody-lysate complex.

• Add ~100 µL of beads per 1 mL of lysate.
• Incubate for ~ 2 hrs at 4°C, with gentle swinging.

Wash the beads three times with 1 mL of pre-cooled TBS to wash the conjugate of beads-antibody-antigen.

• Spin down the tube at the manufacturer's recommended speed for 2 minutes at 4°C.
• Aspirate and discard the solution, keeping the beads in the tube.
• Add ~ 1 mL of pre-cooled TBS to the beads and repeat the steps above.

Stage 4 - Elution

After washing, we’re ready to elute the immune complex from the beads. Elution can be done in various buffer conditions, including glycine (a non-denaturing elution buffer that preserves the protein complex structure), Laemmli (a denaturing buffer that will denature proteins), and urea buffer (a denaturing buffer suitable for mass spectrometry analysis).

Of all these buffers, Laemmli buffer containing SDS is the harshest, as it will also elute non-covalently bound antibodies and antibody fragments along with the protein of interest, and will denature proteins, disrupting protein complexes. In contrast, glycine buffer gently elutes the protein complex with a reduced amount of eluted antibody, preserving its native state.

This method produces a slightly less concentrated sample but keeps the protein in its native state. Since this is a non-denaturing buffer, the beads can also be re-used once the protocol is complete.

Materials required

• 0.1 – 0.2 M Glycine, pH 2.6
• Tris-HCl, pH 8.5
• Your samples bound to beads

Steps

Add an equal volume of glycine to the beads.

• Incubate for 10 mins at room temperature, with gentle agitation.
• The low pH should separate the protein of interest from the antibody beads.

Separate the beads from the solution using a magnetic rack (for magnetic beads) or light centrifugation (for agarose beads).

• For magnetic beads: place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
• For agarose beads: spin down the tube at the manufacturer's recommended speed for 2 mins.
• Aspirate the solution, leaving the beads behind, and transfer this solution to a new tube.

Neutralize the pH of the solution by adding Tris-HCl.

Optional: if not using immediately, aliquot samples, snap freeze, and store at -80°C.

Analyze the IP results using western blot or another technique of your choice.

• Compare the results of IP performed with your capture antibody and isotype control antibody.
• For Co-IP, use an antibody targeting the interacting protein you are interested in rather than an antibody targeting the protein you pulled down.

This method results in a highly concentrated sample that can be loaded directly onto an SDS-PAGE gel for western blot analysis. The main drawback of this method is that it denatures proteins, so it is unsuitable if you need to study the protein in its native state. The buffer and high temperature used will also destroy the beads, so they cannot be re-used.

Materials required

• Denaturing buffer: (example: 0.1 M DTT, 1x LDS, or any other buffer of your choice)
• Your samples bound to beads
• Magnetic rack or microcentrifuge

Steps

Add an equal volume of the denaturing buffer to the beads.

• Incubate for 5 mins at 100°C.

Separate the beads from the solution using a magnetic rack (for magnetic beads) or light centrifugation (for agarose beads).

• For magnetic beads: place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
• For agarose beads: spin down the tube at the manufacturer's recommended speed for 2 minutes.
• Aspirate the solution, leaving the beads behind, and transfer this solution to a new tube.

Optional: if not using immediately, aliquot samples, snap freeze, and store at -80°C.

Analyze the IP results using western blot.

• Compare the results of IP performed with your capture antibody and isotype control antibody.

Materials required

• Pre-urea wash buffer (50 mM Tris, 1 mM EGTA, 75 mM KCl, pH 8.5)
• Urea buffer (6–8 M Urea, 20 mM Tris, 100 mM NaCl, pH 7.5)
• Your samples bound to beads
• Magnetic rack or microcentrifuge

Steps

Wash the beads three times with pre-urea wash buffer.

For magnetic beads:

• Place the tube on a magnetic rack for ~1 minute; the magnet should pull the beads to the bottom of the tube.
• Aspirate and discard the solution, keeping the beads in the tube.
• Add ~ 1 mL of fresh wash buffer to the beads and repeat the steps above.

For agarose beads:

• Spin down the tube at the manufacturer's recommended speed for 2 minutes at 4°C.
• Aspirate and discard the solution, keeping the beads in the tube.
• Add ~ 1 mL of fresh wash buffer to the beads and repeat the steps above.

Add an equal volume of urea buffer to the beads.

• Incubate for 30 mins at room temperature, with gentle agitation.
• The urea buffer will dissociate the protein of interest from the beads.

Separate the beads from the solution.

For magnetic beads:

• Place the tube on a magnetic rack for ~1 minute; the magnet should pull the beads to the bottom of the tube.
• Aspirate the solution, leaving the beads behind, and transfer this solution to a new tube.

For agarose beads:

• Spin down the tube at the manufacturer's recommended speed for 10 minutes.
• Aspirate the solution, leaving the beads behind, and transfer this solution to a new tube.

Optional: if not using immediately, aliquot samples, snap freeze, and store at -80°C.

Analyze the IP results using western blot or another analytical technique.

• Compare the results of IP performed with your capture antibody and isotype control antibody.

Antibody selection and preparation

Choosing the right antibody is a critical factor in the success of immunoprecipitation. The antibody must be highly specific to the target protein to ensure efficient and selective capture of the protein of interest. Monoclonal antibodies, which recognize a single epitope on the target protein, offer high specificity and are ideal for applications where minimizing cross-reactivity is essential. In contrast, polyclonal antibodies recognize multiple epitopes on the same protein, which can enhance the efficiency of protein capture, especially when the target protein is present at low levels or in different conformations. The choice between monoclonal and polyclonal antibodies depends on the experimental goals, the nature of the target protein, and the availability of validated antibodies. Proper preparation of antibodies, including optimizing their concentration and, if necessary, conjugating them to beads, is essential for maximizing the yield and specificity of immunoprecipitation. Using high-quality, well-characterized antibodies ensures that the antibodies recognize the protein of interest effectively, leading to reliable and reproducible results.

Safety precautions

Ensuring laboratory safety is paramount when performing immunoprecipitation experiments. Always wear appropriate personal protective equipment (PPE), including gloves, lab coats, and safety glasses, to protect against exposure to hazardous chemicals and biological materials. Many reagents used in IP, such as lysis buffers, may contain detergents or other chemicals that can be irritants or allergens, so handle them with care and avoid direct contact with skin or eyes. Work in a well-ventilated area, and use a fume hood when handling volatile substances or preparing solutions that may release harmful vapors. Properly dispose of biohazardous waste, including used tips, tubes, and any materials contaminated with cell lysates or lysis buffers, according to your institution’s safety protocols. By following these safety guidelines, you help maintain a safe and healthy laboratory environment for yourself and your colleagues.