Fundamentals of protein affinity chromatography: How to prepare and properly use an affinity matrix

Affinity chromatography (AC) is a powerful method that relies on the principle of molecular recognition, exploiting the ability of various substances and biological compounds to form specific pairs, either reversibly or irreversibly. Attaching one reactive component to a solid-phase substrate allows the capture of a second interacting substance under mild conditions, even from the complex biological materials. When optimal elution conditions are adjusted, specifically captured molecules are quantitatively released from the affinity matrix, achieving them in a high purity. A well-designed affinity matrix combined with optimized separation conditions facilitates the acquisition of bioactive molecules in their native form. Given the wide range of substances suitable for affinity isolation, this article introduces the readers to the basics of protein AC, with the intention to provide a practical guide on how to prepare the affinity matrix and apply it for the effective isolation and purification of protein molecules.