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Protein purification refers to the process of isolating one or more protein molecules from a complex mixture. This can be done for analytical purposes, such as characterization of the structure and functions of a protein, or for preparative purposes, such as purifying monoclonal antibodies. Protein purification exploits a diverse array of physical and chemical properties of biomolecules, including shape, size, charge, hydrophobicity, and binding affinities. Preparative purification yields large quantities of target molecules for further downstream processing, while analytical techniques produce smaller volumes of isolates for research.

Affinity chromatography

Affinity chromatography is one of the most common isolation methods for biomolecules. Also called affinity purification, this process isolates biomolecules from a mixture based on specific and reversible binding interactions between the molecules of interest, found in the mobile phase, and a ligand that is immobilized on a matrix such as a resin or fiber-based material, called the stationary phase.

Biological interactions commonly used in affinity chromatography include antibody and antigen, virus, or cell; metal ions and histidine- (his)-tagged proteins; glutathione and glutathione-S-transferase or GST-tagged proteins; enzyme and substrate analog, inhibitor, or cofactor; or lectin and polysaccharide or glycoprotein.

Affinity chromatography is also used to remove specific contaminants, such as thrombin and Factor Xa.

What happens during affinity chromatography?

During affinity chromatography, a liquid such as a cell lysate containing the molecule of interest is passed over an affinity resin, prepacked column, or fiber-based unit containing the ligand or binding partner of the target molecule. The target molecule binds the ligand and remains bound to the stationary phase until it is eluted.

Affinity resins FAQs

Below are answers to some frequently asked questions about affinity resins.

What are affinity resins?

Affinity resins are matrixes made of agarose or other material coupled to an affinity ligand that binds a molecule of interest.

What is a group-specific affinity resin?

Group-specific affinity resins are for purification of specific groups of molecules, such as glycoproteins and coagulation factors. A group-specific affinity resin has an affinity for a group of related substances rather than for a single type of molecule. The same general ligand can be used to purify several substances (such as members of a class of enzymes) without the need to prepare a new medium for each different substance in the group. Within a group there is either structural or functional similarity. The specificity of a group-specific affinity resin or prepacked column derives from the selectivity of the ligand and the use of selective elution conditions. Affinity resins or columns can be used either for purification or removal of a target substance. In the case of removal, the depleted sample is collected during sample application and wash.

What is the purpose of affinity purification?

Affinity purification is used in a broad range of applications like nucleic acid purification and monoclonal antibody (mAb) purification. Through affinity purification, you can also purify viral vectors like adeno-associated virus (AAV). Other applications of affinity purification include:

  • Downstream plasmid processing
  • Albumin purification
  • Polysaccharide purification
  • Glycolipid purification
  • Enzyme purification
How do I choose an affinity resin for my application or molecule?

You can use Cytiva’s Purify app to select chromatography resins, columns, systems, or accessories.