How does affinity chromatography elute proteins of interest?

Affinity chromatography is a powerful purification technique used to isolate proteins based on their specific interactions with a ligand that is immobilized on a solid matrix inside a column. When proteins are applied to this column, those that have a high affinity for the ligand will bind tightly, while others will wash away.

To elute the proteins of interest, the introduction of free ligand is highly effective. By adding free ligand to the system, it competes with the protein for binding sites on the column. The proteins that are bound to the immobilized ligand will release from the column as they preferentially bind to the free ligand in solution. This process ensures that the target protein is efficiently eluted while other non-specific proteins remain bound to the column until conditions change.

The other methods mentioned—changing salt concentration, altering pH, or applying heat—do not specifically target the interaction between the protein of interest and its ligand in the same precise manner as adding free ligand does. For example, increasing the salt concentration can affect ionic interactions but does not specifically promote the release of a bound protein in the context of affinity chromatography; rather, it could lead to a different class of interactions being disrupted. Similarly, altering pH can affect a protein's charge and solubility but does