Immunoprecipitation, ELISA, antibody purification, and multiplex assays require the attachment of specific antibodies to a solid support such as a column, polystyrene plate, agarose bead, or superparamagnetic nanoparticle. There are a number of ways that antibodies are attached to solid supports. Some of these include:
- covalently bonding the antibody’s primary amines directly to the surface
- biotin-streptavidin affinity linkages
- protein A and G
Direct covalent attachment of an antibody to the support surface is not always an option. It is highly dependent on the material and surface chemistry of the support. Chemical coatings can be applied to the surface to improve binding, but that isn’t always an option. Biotin-avidin affinity chemistry can be a very specific way to attach antibodies, but it has the added steps of biotinylation and avidin tagging. Protein A and G binding is a more general method of antibody functionalization because it works for a variety of IgG antibodies and isn’t antigen-specific. It is an inexpensive method to attach a wide selection of antibodies to solid supports. Magnetic protein A beads are particularly useful for immunoprecipitation protocols utilizing biomagnetic separation to improve the speed and efficiency of the recovery process.
Protein A is a surface protein originally found in the cell wall of Staphylococcus aureus bacteria. Recombinant protein A is commercially harvested from E.coli for practical use. It has four binding sites that bind to the Fc region of the IgG antibody. Human IgM, IgE, and IgA antibodies will not bind to protein A or G. Protein A is very specific to IgG antibodies. For this reason protein A is often used as a method of attaching IgG antibodies to the surfaces of magnetic and agarose beads used for immunoprecipitation. Protein A beads are commercially available for purchase. This eliminates time-consuming steps for the customer. He or she can simply purchase the protein A beads and incubate them with the desired IgG antibody.
IgG antibodies of many species bind to protein A; each has a different affinity depending on species and IgG subtype. Some IgG subtypes don’t bind well at all to protein A beads. This is why another cell wall protein called protein G is also used to functionalize solid support systems with IgG antibodies. The two proteins are similar in that they have special domains with IgG affinity, but their binding specificity differs slightly. Some IgG types which have poor affinity to protein A actually have high affinity for protein G. Sometimes a recombinant A/G protein is used to bind IgG antibodies to a solid support. Information about species-specific IgG subtype affinity to proteins A and G is readily available.
Protein A beads, especially magnetic protein A beads, are a powerful tool in the biosciences because it is essential to be able to identify and purify a protein of interest in order to study it. Biomagnetic separation, in conjunction with magnetic protein A beads, has expanded the toolkit available to accomplish goals in both the research laboratory and in the clinic.