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Affinity Chromatography

How does AFC work, click here for the video!

Affinity Chromatography (AFC) makes use of specific binding interactions between molecules. This specificity makes it attractive as a capture step to isolate the target molecule from crude extracts. A particular ligand is chemically immobilized to a solid support so that when the crude extract is passed over the column, those molecules having a specific binding affinity to the ligand become adsorbed. After the non-specifically binding components are washed away, the target molecule is desorbed from the support, resulting in its purification from the original sample.

In AFC the mechanism of binding and elution varies depending on the immobilized ligand used. In Protein A chromatography elution is accomplished by decreasing the pH. In other cases such as with Chelate resins (for Immobilized Metal Affinity Chromatography or IMAC) the binding and elution mechanism may change through the use of different metals or chemical modifiers.

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