Affinity Chromatography and its use in Antibody Purification
The availability of purified antibodies is essential to various applications. While various purification methods are available, affinity chromatography is the most popular method for antibody purification. Affinity chromatography offers high selectivity, simplicity, and efficiency.
What is Affinity Chromatography?
Affinity chromatography is a laboratory technique that purifies proteins or protein complexes from a biochemical mixture. It relies on the reversible interaction between a protein and a specific ligand immobilized in a chromatographic matrix. Unlike other purification methods, such as gel filtration or size-exclusion chromatography, affinity chromatography manipulates specific molecular properties and binding interactions between molecules to purify the protein of interest.
In affinity chromatography, a particular ligand chemically immobilizes to a solid support. Hence, the molecules with specific binding affinity bind to the ligand after a complex mixture passes over the column. While other sample components wash away, the bound molecule separates from the support (e.g., by changing pH, ionic strength, or adding a competitive ligand), resulting in purification from the original sample. Affinity chromatography is ideally used to purify specific biomolecules or complexes, such as antibodies, enzymes, receptor proteins, and DNA-binding proteins. It offers high selectivity and can yield highly purified samples.
How Does Antibody Purification by Affinity Chromatography Work?
Antibody purification is the selective enrichment or specific isolation of antibodies from serum (polyclonal antibodies), ascites fluid, or cell culture supernatant of hybridoma cell line (monoclonal antibodies).
Antibody Purification is based on the high affinity and specificity of Protein A, Protein G or Chimeric Protein A/G for the Fc-region of lgG from various species. The binding of an antibody to the ligand is reversible, and the antibody is eluted by lowering the pH. In affinity chromatography, the sample is applied under conditions that favor specific binding to the ligand due to electrostatic and hydrophobic interactions. After washing away the unbound materials, the bound protein is recovered by changing the buffer conditions, resulting in its purification from the original sample.
Antibody Purification Resins
Three bacterial proteins, Protein A, Protein G, and Protein L Protein A, Protein G, and Protein L, have been well characterized for their antibody-binding properties. These proteins have been used routinely for affinity purification of key antibody types from a variety of species. Unnecessary sequences are removed from these recombinant proteins, which are, therefore, smaller than their native counterparts. A genetically engineered chimeric form of Protein A and Protein G, called Protein A/G, is also widely available.
G-Biosciences offers a wide selection of resins for of purifying monoclonal and polyclonal antibodies, including Immobilized Protein A, Immobilized Protein G, and Immobilized Protein A/G. These resins bind the constant domains of the protein, allowing for the enrichment of all antibodies from the starting serum or ascites. As stated above, a ligand specific to the antibody of interest needs to be covalently immobilized to a solid support, such as agarose beads. G-Biosciences offers several resins for the immobilization of ligands. The choice is determined by the residues that can be used on the ligand for conjugation and the coupling chemistry. Following coupling chemistry offered by G-Biosciences:
For coupling through primary amines:
For coupling through carboxyl groups:
For coupling through sulfhydryl groups:
For coupling through carbohydrate groups:
G-Biosciences has all the resources for successful antibody purification by affinity chromatography.
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