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Affinity chromatography principles

Affinity chromatography is a common technique for isolating proteins, such as monoclonal antibodies (mAbs) or antibody fragments, from complex mixtures. It relies on specific binding interactions between the target biomolecule and a ligand or binding partner. Protein A is a ligand frequently used to purify mAbs in bioprocessing. In affinity chromatography, an affinity ligand is typically immobilized by coupling to a base matrix such as agarose or electrospun fibers and used as the stationary phase in chromatography. Affinity chromatography may be used to isolate a protein of interest for research, or it may be part of a multistep purification process for biomanufacturing. Affinity chromatography is also used to purify tagged recombinant proteins.

Common antibody affinity ligands

Affinity chromatography requires knowledge of the binding affinity of specific analytes to ensure successful separation protocols. Protein A is a frequently used ligand that has a strong binding interaction with immunoglobulin G (IgG) from many species. Protein G also binds IgG from several species, but with different binding specificities than protein A. Protein L binds only antibodies that contain a kappa light chain and is often used to purify mAbs. 2-Mercaptopyridine has high affinity for IgM.

Protein A columns and antibody affinity FAQs

Here are answers to some frequently asked questions about protein A columns and antibody affinity products:

What is protein A?

Protein A is a cell-wall surface protein of the Staphylococcus aureus bacterium that can bind immunoglobulins (Igs) from various species.

What is protein A chromatography?

Protein A chromatography is used frequently in biomanufacturing. This technique uses protein A coupled to a base matrix, such as a protein A column or resin, to capture an antibody of interest. Protein A has five immunoglobulin-binding domains and is often used for the purification of mAbs for research or preparative purposes.

What are protein A resins?

Protein A resins are affinity chromatography resins in which a base matrix, such as agarose, is coupled to a protein A ligand.

What are protein A columns used for?

Protein A columns are prepacked with a protein A resin for use in affinity chromatography.

What is the difference between protein A and protein G?

Protein A and protein G have structural similarities but slightly different affinities for different IgG subclasses and for IgG from different species. Both are used in affinity chromatography. Our Purify app can help you choose which one would be best for your molecule.

What are kappa and lambda chains?

Immunoglobulins typically are made of two heavy chains and two light chains. Kappa chains and lambda chains are two types of Ig light chains. Cytiva offers protein L chromatography resins for affinity capture of antigen-binding antibody fragments (Fabs) containing kappa light chains and products for affinity capture of human lambda Fabs.