Mass Spectrometry & Purification Techniques
Open Access

Affinity Purification and its Types

Mingming Jia^{* *}Correspondence: Mingming Jia, Department of Chemistry, University of Uyo, Uyo, Nigeria, Email:

Author info »

Description

To separate a protein of interest from other proteins and components in a crude cell lysate or other sample, various methods are used. The most effective of these methods is affinity chromatography, also known as affinity purification, in which the protein of interest is purified based on its specific binding properties to an immobilized ligand. A variety of methods can be used to separate proteins and other macromolecules of interest from crude extracts or other complex mixtures. The most basic method for separating one type of macromolecule from another is selective precipitation.

However, the majority of purification methods involve some form of chromatography, in which molecules in solution (mobile phase) are separated based on differences in chemical or physical interaction with a stationary material (solid phase). Gel filtration (also known as size-exclusion chromatography or SEC) separates molecules based on their size using a porous resin material (i.e., physical exclusion). Ion exchange chromatography separates molecules based on the strength of their overall ionic interaction with a solid phase material (i.e., nonspecific interactions).

Affinity chromatography (also known as affinity purification), on the other hand, makes use of specific binding interactions between molecules. When a complex mixture is passed through the column, those molecules with specific binding affinity to the ligand become bound. Following the removal of other sample components, the bound molecule is stripped from the support, resulting in its separation from the original sample.

Types of affinity purification

For the development of a successful procedure, different classes of affinity targets, as well as different purification goals, necessitate consideration of different priorities (e.g., high purity versus. high yield), technical constraints, and buffer conditions.

Antibody purification: Affinity purification techniques are used in several antibody purification methods. Small volumes of serum, ascites fluid, or culture supernatant are typically used in laboratory-scale antibody production. The antibody must be partially or fully purified depending on how it will be used for various assay and detection methods. Ammonium sulphate precipitation this method separates total immunoglobulin from other serum proteins.

Antigen purification using antibodies: Specific antibodies are most commonly used in assays to detect antigens of interest, but they can also be used to purify antigens. Because specific antibodies are expensive to produce or obtain commercially, this method is rarely used for large-scale antigen purification. Instead, it is almost entirely used on very small scales, most notably in immunoprecipitation assays. When purified antibody is available, it can be covalently immobilized to beaded agarose or other affinity supports using any of several efficient conjugation chemistries. Covalent immobilization via primary amines, such as Thermo Scientific AminoLink Plus coupling kits, is a particularly simple and efficient method for preparing an antibody affinity column.

Immunoprecipitation and co-immunoprecipitation: Immunoprecipitation (IP) is the affinity purification of antigen on a small scale using a specific antibody. Traditional immunoprecipitation involves binding an antibody-antigen complex with immobilised Protein A or G agarose resin (Protein A or G binds the antibody, which is bound to its antigen), then purifying the antigen in sample loading buffer for gel electrophoresis. Co-immunoprecipitation (Co-IP) entails attempting to capture and detect not only the direct antigen but also any proteins in the lysate mileu that are interacting (i.e., bound to) the antigen. This purification scheme uses three levels of affinity interaction in the traditional format with Protein A or Protein G.

Fusion tag protein purification: When proteins are expressed recombinant, extra amino acids, a functional domain, or the entire protein is frequently appended to aid in purification and manipulation. These fusion tags are added to the DNA that encodes the native protein sequence and are added to the recombinant protein. A short string of six to nine histidine residues that binds to metal ions such as nickel or cobalt is one of the most common fusion tags. Glutathione S-transferase (GST) is another fusion tag that binds tightly to reduce glutathione.