Affinity chromatography is a chromatographic technique that uses the specific affinity between biomolecules for separation. Specifically, affinity chromatography involves immobilizing a ligand on an inert carrier, and the target protein in the mixture is specifically bound to the ligand and thus captured, while the extraneous impurity components are washed off with the mobile phase, and then a suitable eluent is selected to elute the target protein from the carrier, resulting in a single component of purified material. Recombinant protein A/G/L (Protein A/G/L) is widely used for the isolation and purification of antibodies. Each recombinant protein has specific affinity and specificity, and the appropriate recombinant protein can be selected to purify different kinds of antibodies.
Protein A
Protein A, 42 kDa in size, is a surface protein encoded by spa gene and derived from the cell wall of Staphylococcus aureus, which binds specifically to immunoglobulins and is widely used for antibody detection and purification. Natural protein A is structurally stable, acid- and base-intolerant, has no disulfide bonds within the molecule, and has five highly homologous regions that bind the Fc fragment of IgG. Protein A has a strong affinity for IgG from a variety of sources and does not affect the activity of IgG-bound antigens. Also, non-IgG antibodies such as IgA, IgM or IgE have the ability to bind to Protein A. Currently, affinity chromatography media with Protein A as the affinity ligand have been widely used for the purification of monoclonal antibodies and Fc recombinant fusion proteins.
Protein A, Ig-binding domain
Protein G
Protein G has a molecular size of approximately 60 kDa and is a C and G streptococcal cell surface protein. Protein G binds IgG, but does not have similar binding reactions with IgA, IgM, IgD, and IgE. Protein G can bind more extensively, more strongly, and to more types of IgG than Protein A, but at a lower load. Natural Protein G binds not only to the constant region of immunoglobulins, but also to albumin and α2-macroglobulin, which makes it impossible to remove albumin and macroglobulin present in the system by Protein G affinity chromatography purification of antibodies.
Protein L
Protein L was originally isolated from the surface of Peptostreptococcus magnus and was named after the immunoglobulins that were found to bind through L-chain interactions. Protein L binds mainly to the κ light chain with five small homologous structural domains at the N-terminal end of the binding site. Protein L binds IgG, IgM, IgA, IgE and IgD, as well as single-chain antibodies ScFv and Fab fragments. In addition, Protein L does not interfere with the antigen-binding site of the antibody, allowing it to be used in immunoprecipitation assays, even with IgM.
Protein L b1 domain
In general, the choice of Protein A/G/L will depend on the subtype and grade of the antibody, as well as the needs of specific applications. Creative Biolabs' experienced scientists have built a professional platform to provide customized non-IgG antibodies production and purification service for customers in need. Services beyond standard procedures will surely meet customers' research and development needs.
Reference
- "Protein A". Wikipedia, Wikimedia Foundation, 15 Feb. 2023.
- "Protein L". Wikipedia, Wikimedia Foundation, 15 Feb. 2023.
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