Protein A and Protein G are bacterial cell wall proteins that have primary binding sites for human and mammalian immunoglobulin G (IgG) antibodies. Protein G was first isolated from Streptococcal bacteria strains C and G. Similarly, protein A was originally found on the cell wall of the bacteria Staphylococcus aureus. These proteins have primary binding domains for the Fc region of (IgG) antibodies, but can also recognize the Fab region of certain IgG subclasses. For the bacteria this is useful because binding IgG’s at the Fc region prevents macrophages from recognizing them, which in turn prevents phagocytosis of the invading bacteria by the host immune system.
Antibody structure and classes (isotypes)
There are 5 main classes of immunoglobulin antibodies: IgG, IgD, IgE, IgA, and IgM. They differ in the type of heavy chain and in their ability to join together to form higher order structures. The basic structure of an immunoglobulin is a Y-shape. The base of the Y is called the Fc region, while the two extensions of the Y are called the Fab regions. The Y itself is composed of two heavy chains and two light chains that are held together by a combination of non-covalent interactions and disulfide chains. The two tips of the Y are the variable regions where the antigen binding sites are located. This basic Y structure is usually the only form that IgG, IgD, and IgE antibody classes are found in. However, IgA can be a combination of two Y’s, and IgM is a pentamer.
Protein A vs Protein G
This ability of protein A and G to specifically recognize IgG antibodies makes them a useful tool. As such, they are often used commercially to purify IgG antibodies or in biosensor development for the oriented immobilization of IgG’s to a sensor surface for antigen capture. Protein A and G are structurally very similar, but they have slightly different affinities for IgG subclasses across different species. These affinities overlap, but in general, protein A has greater affinity for rabbit, pig, dog, and cat IgG whereas protein G has greater affinity for mouse and human IgG. This, of course, is just a general guideline. Refer to affinity tables for detailed information for each IgG subclass in each species when carefully comparing protein A vs protein G for experimental design.
Recombinant vs native IgG binding proteins
Native protein A and G have albumin binding sites and cell wall binding regions in addition to IgG recognition sites. To prevent nonspecific binding of these proteins during bioassays, scientists have engineered recombinant protein A and G that lack the cell wall and albumin binding regions. These recombinant proteins are harvested from E. coli cultures. Recombinant protein A has 5 IgG binding domains, which is more than the native protein A. Therefore, recombinant protein A has increased affinity for IgG than native protein A. In addition, recombinant protein A/G has been developed. Protein A/G contains 4 protein A binding domains and two protein G binding domains, which broadens the subclasses of IgGs to which it has affinity.
- Antibody affinity
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