Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. It enables the purification of a biomolecule on the basis of its biological function or individual chemical structure.
Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity.
Successful affinity purification requires a biospecific ligand that can be covalently attached to a chromatographic matrix. The coupled ligand must retain its specific binding affinity for the target molecules and, after washing away unbound material, the binding between the ligand and target molecule must be reversible to allow the target molecules to be removed in an active form. Any component can be used as a ligand to purify its respective binding.
1. Affinity medium is equilibrated in binding buffer.
2. Sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes
through the column.
3. Target protein is recovered by changing conditions to favour elution of the bound molecules. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Target protein is
collected in a purified, concentrated form.
4. Affinity medium is re-equilibrated with binding buffer.
Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity.
Successful affinity purification requires a biospecific ligand that can be covalently attached to a chromatographic matrix. The coupled ligand must retain its specific binding affinity for the target molecules and, after washing away unbound material, the binding between the ligand and target molecule must be reversible to allow the target molecules to be removed in an active form. Any component can be used as a ligand to purify its respective binding.
1. Affinity medium is equilibrated in binding buffer.
2. Sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes
through the column.
3. Target protein is recovered by changing conditions to favour elution of the bound molecules. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Target protein is
collected in a purified, concentrated form.
4. Affinity medium is re-equilibrated with binding buffer.
No comments:
Post a Comment