Immunoprecipitation Antibodies

Immunoprecipitation (IP) is a technique that uses bead-bound antibodies for extracting proteins from cell or tissue lysates. It is performed in one of two main ways, the most common of which involves incubating the antibody with the beads before adding the antibody-bead complex to the sample. Once the target of interest has been captured, the beads are pelleted from solution and washed several times, then the protein is eluted with an appropriate buffer. Alternatively, when isolating scarce targets or working with low-affinity antibodies, the antibody may be incubated with the sample prior to bead addition. The eluted proteins are then typically analyzed by western blotting. The types of beads that are used for immunoprecipitation are either agarose or magnetic. To enable antibody binding, they are commonly functionalized with Protein A, Protein G, or Protein A/G (a recombinant fusion protein that contains binding domains from both proteins), although beads that have been functionalized with streptavidin or an anti-species antibody are also popular. While agarose beads have historically been favored for IP, magnetic beads are now preferred as they have a more uniform size and shape, which can improve experimental reproducibility. Magnetic beads also benefit from shorter incubation times and gentler (centrifugation-free) washing, which can help to prevent protein damage or accidental sample loss. When selecting antibodies for immunoprecipitation, it is important to consider the host species, as well as the antibody isotype and subtype, since the binding capacities of Protein A and Protein G vary based on these characteristics. It is also essential to select antibodies that are validated for the IP application, and thus known to recognize the native protein. If such an antibody is not available, it may be worth testing a product that has been validated for another application involving native protein detection, such as immunohistochemistry (IHC) or flow cytometry, using our trial size antibodies. An additional factor to consider is whether to use a monoclonal or a polyclonal antibody for immunoprecipitation. Polyclonal antibodies are often used for co-immunoprecipitation (co-IP) experiments, where the target of interest is captured in complex with other proteins. Unlike monoclonal antibodies, which recognize just a single epitope, polyclonal antibodies exhibit epitope redundancy, which may increase the likelihood of target capture if certain epitopes are blocked as a result of complex formation. Western blot detection of IP samples can be challenging if the target of interest shares a similar molecular weight to antibody heavy or light chains (50 kDa and 25 kDa, respectively). This is because the capture antibody is released from the beads during the elution step, giving rise to antibody heavy and light chains that can be detected with secondary antibody reagents. Ways of avoiding this include using labeled primary antibodies for detection, or selecting secondary antibodies that recognize only intact immunoglobulins.