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How Does Immunoprecipitation Work?
Immunoprecipitation (IP) is a technique for small-scale purification of proteins from cell or tissue lysates. It can be used to enrich a single protein, allowing for investigation of its expression, activation, or modification state in different sample types. In addition, variations of IP have been developed for studying interactions between a target of interest and other proteins or nucleic acids.
How does IP work?
IP works by using a target-specific antibody to capture a protein in solution, enabling its extraction through antibody binding to functionalized agarose or magnetic beads. Typically, the antibody and beads are incubated together before being mixed with the sample; the beads are then pelleted via centrifugation or using a magnet, washed several times, and the protein eluted with a suitable buffer.
However, in situations where the target protein is present in only low concentrations, or the antibody has a weak affinity for the antigen, it is more common for the antibody to be mixed with the sample prior to incubation with the beads.
What do I need to consider for antibody selection?
Because IP is used for isolating native proteins, it is essential to select an antibody that recognizes the protein in its natural form. If IP validation data is not available for a particular product, antibodies that have been proven to work for immunohistochemistry (IHC) may be worth a try.
For co-immunoprecipitation applications (see below), polyclonal antibodies are often favored over monoclonals since, by binding multiple epitopes, they are more likely to capture the target if a particular site is blocked by another protein within the same complex.
Our product offering includes over 450 antibodies that have been validated for IP and our non-specific mouse, rat, rabbit, and goat antibodies are widely literature cited.
How do I choose between agarose and magnetic beads?
IP was originally developed using agarose beads, which have a porous structure that provides a high level of antibody binding. Yet, agarose beads are now recognized to present inherent challenges, not least due to their non-uniform size and shape, which can compromise the reproducibility of experimental results.
Other limitations of agarose beads include long incubation steps (to enable antibody diffusion) and a requirement for extensive, centrifuge-based washing, which risks causing protein damage or accidental sample loss. For these reasons, magnetic beads, which have a consistent size, shorter incubation times, and more gentle wash steps have largely replaced agarose beads as the preferred IP support.
Further advantages of using magnetic beads for IP are that they circumvent the need for pre-clearing (the process of incubating all of the reaction components together, but using a non-specific antibody from the same host species as the IP antibody to prevent unwanted protein capture during the IP reaction) and increase opportunities for introducing automation.
How should the beads be functionalized?
Beads coupled to either Protein A or Protein G are widely used for IP. But, since Protein A and G exhibit different binding capacities for different antibody species, classes, and subclasses, it is recommended that researchers consult a table of antibody-binding characteristics when identifying a suitable protein for their particular immunoprecipitating antibody.
Alternatively, beads are available coupled to Protein A/G (a recombinant protein with four Protein A and two Protein G antibody binding sites), which binds all of the antibody types that each individual protein binds.
Where researchers wish to avoid the need for Protein A/G-dependent antibody immobilization altogether, options include streptavidin-coated beads (for use with biotinylated antibodies) and beads that have been coupled to anti-species antibodies.
How do I elute the bound protein?
Because IP reactions are usually analyzed by Western blotting, elution often involves little more than heating the beads in sample loading buffer to release and denature the bound proteins. A major issue with this approach is that both the target protein and the capture antibody end up in solution.
If the target of interest shares a similar molecular weight to antibody heavy (50 kDa) or light (25 kDa) chains, the presence of these proteins on the resultant blot can obscure its detection with secondary antibody reagents. One way of avoiding this issue is to use secondary antibodies that recognize only native primary antibodies for detection.
Another is to use heavy chain-specific secondary antibodies for detecting proteins with a molecular weight of around 25 kDa and light chain-specific antibodies for detecting 50 kDa proteins. Secondary antibody use can be eliminated by using labeled primary antibodies. Where another type of analysis will be performed, the elution buffer should be tailored accordingly.
We offer heavy chain-specific and light chain-specific secondary antibodies for species including mouse, human, rat, rabbit, goat, monkey, and chicken, as well as an extensive selection of labeled primary antibodies.
What are some variations of IP?
As well as being used to pull down individual proteins, IP has been adapted for several other applications. Co-immunoprecipitation (co-IP) is identical to IP, except it is used for capturing a protein of interest in combination with its binding partners, or in complex with other proteins.
Chromatin immunoprecipitation (ChIP) assays are instead used for identifying which regions of the genome are targeted by DNA binding proteins; following IP-based capture of the target protein, the DNA is released for sequencing. RNA immunoprecipitation (RIP) is similar to ChIP, but captures RNA-binding proteins, enabling RNA identification by RT-PCR and cDNA sequencing.
