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Protein purification is a series of processes used to isolate one or more native or recombinant proteins from complex mixtures like cells and tissues. The purification of proteins facilitates characterizations of structure and functions of different proteins, and it allows production of therapies. These processes take advantage of different physico-chemical properties like shape, size, ionic charge, specific binding, and hydrophobic groups. Protein purification is either analytical or preparative. Analytical purification yields moderate amounts of isolates for research, while preparative purification produces large volumes of purified proteins, often for use as biologics.

Affinity chromatography for protein purification

You can purify proteins and other macromolecules from complex mixtures and crude extracts through a variety of methods. One method is affinity chromatography, in which biomolecules are separated from a mixture based on a specific macromolecular binding interaction.

Affinity chromatography for protein purification takes advantages of binding interactions between a protein of interest and a ligand. The method offers high selectivity and resolution. During affinity chromatography, recombinant protein mixtures pass over a chromatography column that contains a resin with an attached ligand. The ligand selectively binds proteins with high affinity for the ligand. Many contaminants get washed away using buffers, and the bound protein is then eluted with high purity using an elution buffer.

Affinity chromatography purification FAQs

Below are answers to some frequently asked questions about affinity chromatography for protein purification and purification of other molecules.

What is affinity chromatography?

Affinity chromatography is a separation technique that uses the specific binding interactions between an immobilized ligand and its binding partner. It is often a first step in a purification scheme, as high purities can be achieved.

What is affinity chromatography used for?

Affinity chromatography is used in a diverse range of applications, such as monoclonal antibody (mAb) purification, purification of other proteins, and nucleic acid purification. This separation technique also allows use in the purification of viral vectors, such as adeno-associated virus (AAV) for gene therapy. AAV comes in a diverse array of serotypes to allow targeting of different types of tissue; different serotypes will likely differ in their affinity to a particular ligand. You can also utilize affinity purification for the downstream processing of plasmids. Other molecules that can be purified using affinity methods include albumins, enzymes, glycoproteins, polysaccharides, and glycolipids.

How do you purify proteins using affinity chromatography?

Affinity chromatography for protein purification relies on the interaction between a protein and specific ligand. It involves the separation of molecules in solution through binding interaction differences with a ligand in the stationary phase. The affinity of the target protein to the ligand is so high that other molecules bind nonspecifically or not at all. Binding of proteins to ligands attached to a matrix is reversible either by competition or by decreasing affinity using pH and ionic strength.

How do I find the right affinity resin to purify my molecule type?

If your protein is a monoclonal antibody, protein A-based resins are commonly used. Other affinity ligands are also possible, depending on the characteristics of your molecule or fragment. If your molecule is a recombinant protein onto which you’ve engineered an affinity tag, such as polyhistidine or glutathione S-transferase (GST), purify using tagged protein affinity resins. There are many options for other proteins and other types of molecules. Cytiva’s Purify app will guide you through the process for identifying a suitable resin.