Secondary Antibodies for Immunoprecipitation (IP)

Immunoprecipitation (IP) is a technique which uses bead-bound antibodies for extracting a protein analyte from a cell lysate, tissue homogenate or culture supernatant. It can be configured in several different ways, all of which involve forming an analyte-antibody-bead complex before eluting the target of interest for detection, usually via western blot. Key protocol variables include the types of beads that are used (agarose or magnetic), how the beads are functionalized to enable antibody binding, and whether the western blot will employ direct or indirect detection. Most IP reactions use beads that are functionalized with Protein A or Protein G. These naturally-occurring biomolecules bind with varying affinities to the Fc region of different antibody species and subtypes, serving to orient the antibody such that the variable regions are facing outward from the bead. It is also possible for IP reactions to use beads functionalized with Protein A/G, a recombinant fusion protein that was developed to bind all of the antibody species and subtypes recognized by Protein A and G individually, thus simplifying reagent selection. Other bead types used for IP include beads functionalized with streptavidin, which bind to biotinylated antibodies, and those functionalized with anti-species antibodies, which effectively introduce an additional antibody layer into the analyte-antibody-bead complex. Western blot detection of the protein analyte can be challenging when the target of interest has a similar molecular weight to antibody heavy (50 kDa) or light (25 kDa) chains. This is because the elution step releases not only the analyte from the beads, but also the antibody that was used for its capture. A consequence of this is that the antibody is transferred to the membrane along with the protein of interest, where it can be detected by any secondary antibodies used for western blotting. One way of circumventing this issue is to use labeled primary antibodies for direct detection, an approach which also offers the advantage of fewer protocol steps compared with indirect detection. Another option is to use secondary antibodies that recognize only native (intact) IgG molecules, which bind the primary antibodies used for western blot detection but not those involved in the IP reaction.