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Affinity Chromatography
When it comes to separation and analysis of proteins with respect to their specific
and natural properties, affinity chromatography has found wide application. In
1968, the technique was initially used for purification of enzymes and has since
been extended for purification of other bio molecules such as receptor proteins,
immunoglobulin’s, glycoprotein, nucleotides, nucleic acids and even to whole cells
and cell fragments (Wilson and Walker 2010).
Unlike most other forms of chromatography, this chromatographic technique is
most specific as it does not rely on the than physico-chemical properties of analytes.
Rather, it is based on strong specific biological ligand-protein interaction (Narayanan
1994). These interactions not only depend on general properties such as isoelectric
point or hydrophobicity, but on selective properties such as the interactions between
antigen and antibody, enzyme and substrate analogue, nucleic acid and binding proteins as well as hormone and receptors (Fanali et al. 2017).
This technique thus finds wide application in preserving immunological and biological activity of isolated proteins and antibodies. Theoretically it is capable of
giving very high purification, even from complex mixtures, in just one simple process step.
Principle and Method
The technique involves reversible binding of protein of interest to a specific ligand
coupled to column matrices. In general, immobilization of ligand occurs in
three steps:
• Activation of matrix for its reaction with the functional group of the ligand.
• covalently coupling of ligand through some chemical reaction
• Finally, blocking of residual unreacted groups by an excess of a suitable low
molecular weight substance such as ethanolamine.
This provides a higher degree of certainty that all binding will be between the
ligand and the sample. Since only the intended protein is adsorbed from the extract
passing through the column. It is retained in column by making a complex with
ligand, other substances will be washed away. The interaction of ligand-protein can
be reversed, and bound proteins can leave the column either specifically using a
competitive ligand, or non-specifically by decreasing the pH of the buffer, increasing ionic strength or polarity to elute the target protein in bioactive form. This technique is able to concentrate dilute amounts of the expensive molecule, purification
of theraupeutic products and stabilize the protein when adsorbed onto a ligand for
which it has a natural affinity.
Another important feature of affinity chromatography is that proteins often can
be separated from denatured or functionally different forms, since the technique
relies on functional properties. One form of affinity chromatography uses Protein A
M. Manzoor et al.
Affinity Chromatography
When it comes to separation and analysis of proteins with respect to their specific
and natural properties, affinity chromatography has found wide application. In
1968, the technique was initially used for purification of enzymes and has since
been extended for purification of other bio molecules such as receptor proteins,
immunoglobulin’s, glycoprotein, nucleotides, nucleic acids and even to whole cells
and cell fragments (Wilson and Walker 2010).
Unlike most other forms of chromatography, this chromatographic technique is
most specific as it does not rely on the than physico-chemical properties of analytes.
Rather, it is based on strong specific biological ligand-protein interaction (Narayanan
1994). These interactions not only depend on general properties such as isoelectric
point or hydrophobicity, but on selective properties such as the interactions between
antigen and antibody, enzyme and substrate analogue, nucleic acid and binding proteins as well as hormone and receptors (Fanali et al. 2017).
This technique thus finds wide application in preserving immunological and biological activity of isolated proteins and antibodies. Theoretically it is capable of
giving very high purification, even from complex mixtures, in just one simple process step.
Principle and Method
The technique involves reversible binding of protein of interest to a specific ligand
coupled to column matrices. In general, immobilization of ligand occurs in
three steps:
• Activation of matrix for its reaction with the functional group of the ligand.
• covalently coupling of ligand through some chemical reaction
• Finally, blocking of residual unreacted groups by an excess of a suitable low
molecular weight substance such as ethanolamine.
This provides a higher degree of certainty that all binding will be between the
ligand and the sample. Since only the intended protein is adsorbed from the extract
passing through the column. It is retained in column by making a complex with
ligand, other substances will be washed away. The interaction of ligand-protein can
be reversed, and bound proteins can leave the column either specifically using a
competitive ligand, or non-specifically by decreasing the pH of the buffer, increasing ionic strength or polarity to elute the target protein in bioactive form. This technique is able to concentrate dilute amounts of the expensive molecule, purification
of theraupeutic products and stabilize the protein when adsorbed onto a ligand for
which it has a natural affinity.
Another important feature of affinity chromatography is that proteins often can
be separated from denatured or functionally different forms, since the technique
relies on functional properties. One form of affinity chromatography uses Protein A
M. Manzoor et al.