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Protein purification

Protein purification is the isolation of proteins from complex mixtures or crude sources. It can be used to analyze protein structures, functions, or interactions, and for preparative purposes, such as production of therapeutic antibodies. Purification processes make use of variations in physico-chemical attributes, such as biological activity, binding affinity, or size to separate a protein of interest from other molecules in a mixture. Commonly used purification techniques include affinity chromatography, size exclusion chromatography, and hydrophobic interaction chromatography.

Affinity chromatography

Affinity chromatography is a type of liquid chromatography that relies on the specific and reversible binding of an antibody or other protein to an affinity ligand. Ligands are coupled to a base matrix, such as a bead-based resin or electrospun fibers, which serves as the stationary phase in liquid chromatography. A mixture, or mobile phase, containing the biomolecule of interest then flows over the stationary phase. The ligand binds and retains the target molecule as other molecules in the mixture are eluted in a series of wash steps.

Affinity tags

Some proteins, such as monoclonal antibodies, can be purified based on their binding to ligands such as protein A. Purification of other proteins may require fusion of the protein with an affinity tag, a peptide sequence that binds a specific ligand. Once isolated, tagged proteins may be treated with an enzyme or other chemical agent to remove the affinity tag. Some protein tags commonly used in biomanufacturing and research include histidine, glutathione S-transferase (GST), and Strep-tag™ II.

Tagged proteins FAQs

Below are answers to some frequently asked questions about purifying tagged proteins.

What are affinity-tagged proteins?

Affinity-tagged proteins have been fused to an affinity tag such as histidine, GST, or Strep-tag™ II to facilitate their efficient purification.

How do you elute GST-tagged proteins?

Purification of GST-tagged proteins relies on the affinity of GST to glutathione, which in chromatography is coupled to a matrix such as Sepharose™ resin. GST-tagged proteins captured on an affinity matrix are eluted in an elution buffer containing reduced glutathione. Cytiva offers a GST buffer kit for binding, washing, and elution of GST-tagged proteins.

What is a his-tagged protein?

A histidine-tagged (his-tagged) protein has been fused with a string of polyhistidine residues for efficient purification and detection.

How can I choose the right column or resin for my affinity-tagged protein?

Cytiva’s Purify app can help you choose the right prepacked column or resin for purifying your target protein.