Which chromatography method relies on binding interactions to separate proteins?

Affinity chromatography is based on specific binding interactions between a target protein and a complementary ligand that is immobilized on the chromatography matrix. This technique allows for the selective separation of proteins based on their affinity for the ligand. For example, if a protein of interest has a known binding partner, that partner can be attached to a solid surface. When the crude protein mixture is passed through the column, only the protein that interacts with the ligand will bind, while others will be washed away. Afterward, the bound protein can be eluted by using a solution that competes with the binding interactions or alters the conditions to release the protein.

In contrast, size exclusion chromatography separates proteins based on their size, allowing smaller molecules to enter the pores of the resin and delaying their elution compared to larger molecules. Ion exchange chromatography utilizes the charge properties of proteins, separating them based on their net charge at a particular pH. Reverse-phase HPLC separates proteins based on their hydrophobicity, using a non-polar stationary phase and a polar mobile phase to attract more hydrophobic molecules. While all these methods are effective for protein separation, affinity chromatography is unique in its reliance on specific binding interactions, making it particularly powerful for purifying proteins of interest.