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What is affinity chromatography?
Affinity chromatography is a purification technique that typically offers purities >95% in one step.
It makes use of a specific native or added property of the target molecule to isolate it from all contaminants in the sample.
Binding sites of receptors and antibodies or active sites of enzymes are examples of very specific properties that can be used for affinity chromatography. Prosthetic groups like polysaccharides are also used, but then for group separations. Group separations can also be accomplished by using ligands with broader specificity.
Common for all types of affinity chromatography is that a ligand (affinity ligand) specific for the binding site of the target molecule, is coupled to an inert chromatography matrix.
Under binding conditions this chromatography matrix will bind molecules according to its specificity only. All other sample components will pass through the chromatography medium unadsorbed. After a wash step the adsorbed molecules are released and eluted by changing the conditions towards dissociation or by adding an excess of a substance that displaces the target molecule from the affinity ligand.
With recombinant produced proteins a special type of affinity chromatography can be applied thanks to gene fusion techniques.
Special vectors are used to introduce an affinity tag into the target protein by gene fusion. Very often a cleavage recognition site is also introduced between the target protein and the affinity tag. When necessary the affinity tag can then be removed by the aid of a protease specific for this site after purification.
Gradient elution is normally not necessary in affinity chromatography and very simple equipment like a syringe or a peristaltic pump can be used.
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