Affinity chromatography
The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid[1] binding interactions are frequently exploited for isolation of various biomolecules.
Affinity chromatography is useful for its high selectivity and resolution of separation,[2][3] compared to other chromatographic methods.
[5] Types of binding interactions commonly exploited in affinity chromatography procedures are summarized in the table below.
Affinity columns can be eluted by changing salt concentrations, pH, pI, charge and ionic strength directly or through a gradient to resolve the particles of interest.
[12][9][13] Briefly, they are (generalized) activated/functionalized that work as a functional spacer, support matrix, and eliminates handling of toxic reagents.
The protein can then be covalently coupled to a solid support such as agarose and used as an affinity ligand in purifications of antibody from immune serum.
This cysteine residue contains a sulfhydryl functional group which allows the peptide to be easily conjugated to a carrier protein (e.g. Keyhole limpet hemocyanin (KLH)).
The same cysteine-containing peptide is also immobilized onto an agarose resin through the cysteine residue and is then used to purify the antibody.
[15] Immunoaffinity chromatography with monoclonal antibodies immobilized on monolithic column has been successfully used to capture extracellular vesicles (e.g., exosomes and exomeres) from human blood plasma by targeting tetraspanins and integrins found on the surface of the EVs.
[16][17] Immunoaffinity chromatography is also the basis for immunochromatographic test (ICT) strips, which provide a rapid means of diagnosis in patient care.
Methods used to elute the protein of interest include changing the pH, or adding a competitive molecule, such as imidazole.
Histidine tags have an affinity for nickel, cobalt, zinc, copper and iron ions which have been immobilized by forming coordinate covalent bonds with a chelator incorporated in the stationary phase.
For elution, an excess amount of a compound able to act as a metal ion ligand, such as imidazole, is used.
Lectins, such as concanavalin A are proteins which can bind specific alpha-D-mannose and alpha-D-glucose carbohydrate molecules.
Some common carbohydrate molecules that is used in lectin affinity chromatography are Con A-Sepharose and WGA-agarose.
[25] Although there are various ways to perform lectin affinity chromatography, the goal is extract a sugar ligand of the desired protein.
p-aminobenyl-1-thio-β-D-galactopyranosyl agarose is used as the affinity matrix because it contains a galactopyranosyl group, which serves as a good substrate analog for E. coli β-Galactosidase.
This property allows the enzyme to bind to the stationary phase of the affinity matrix and β-Galactosidase is eluted by adding increasing concentrations of salt to the column.
Clinical adaptations have applied this type of chromatography for use in determining long term assessment of diabetic patients through analysis of their glycated hemoglobin.
[30][31] WAC is an affinity-based liquid chromatographic technique that separates chemical compounds based on their different weak affinities to an immobilized target.
Affinity chromatography is part of a larger suite of techniques used in chemoproteomics based drug target identification.
