Protein Purification by Affinity Chromatography

Affinity Chromatography Definition

What is Affinity Chromatography

Affinity chromatography relies on the specific and reversible binding of a protein to a matrix-bound ligand. The ligand can bind directly to either the protein of interest or a tag that is covalently attached to the protein. Affinity chromatography is often the most robust purification procedure and is typically used in the early stages of the purification scheme. This specific affinity interaction is able to capture the target while removing contaminants or other molecules in a solution and in a single step enrich or purify the targeted molecule away from all other molecules that cannot bind the ligand.

The affinity approach is limited to proteins that have a specific binding property, except that proteins are theoretically able to be purified by immunoaffinity chromatography, which is the most specific of all affinity techniques.

Ni-NTA Chromatography Purification

Ni-NTA Agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a His tag. Histidine residues in the His tag bind to the vacant positions in the coordination sphere of the immobilized nickel ions with high specificity and affinity. Cleared cell lysates are loaded onto the matrices. His-tagged proteins are bound, and other proteins pass through the matrix. After washing, His-tagged proteins are eluted in buffer under native or denaturing conditions(See more about His Tag Purification).

Other Common-used Affinity Tags by Chromatography Purification

What is affinity tag? Affinity tag refers to a short peptide added to either the N- or C-end of a recombinant protein to facilitate purification of the expressed protein and the affinity tag sequence usually contains from several to hundreds of amino acids. Here list the most common-used affinity tags by affinity chromatography.

Affinity Chromatography Steps

In affinity chromatography, proteins are loaded on the column under conditions that influence binding between the protein (or tag) and its ligand. The bound protein is washed under conditions that do not disrupt the specific interaction, but that can disrupt any non-specific interactions between contaminating proteins and the stationary phase. The bound protein is then eluted with a buffer containing a competing molecule or conditions that disrupt all protein/protein interactions. Competing molecules bind to the ligand, displacing the protein of interest. This competing molecule is typically removed from the protein of interest either through another chromatographic procedure or dialysis.

Affinity Chromatography Ligand and Conditions

Protein to Purify Ligand Elute With
Antibody (antigen-specific) Antigenic peptide Free peptide
Polyhistidine-tagged protein Ni2+ or Co2+ Imidazole or free histidine
FLAG-tagged protein FLAG-specific antibody FLAG peptide or low pH
GST-tagged protein Reduced glutathione Free glutathione
Myc-tagged protein Myc-specific antibody Low pH
Antibody (class-specific) Protein A , G, or L or protamine Extremes in pH
DNA-binding protein Heparin High ionic strength


Adamikova J, et al. (2019) Chromatographic purification of recombinant human erythropoietin. Biotechnol Lett 41(4-5): 483-493.
Joshi H, et al. (2017) Novel method to rapidly and efficiently lyse escherichia coli for the isolation of recombinant protein. Anal Biochem 528: 1-6.

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