What is the role of imidazole in the purification of His-tagged GFP?

Imidazole plays a critical role in the purification of His-tagged proteins, including Green Fluorescent Protein (GFP). In this process, His-tagged proteins are often purified using affinity chromatography, where the His-tag binds to nickel ions (Ni²⁺) that are immobilized on a resin. The presence of imidazole in the purification buffer is strategically important because it competes with the histidine residues of the His-tag for the nickel binding sites.

As the concentration of imidazole increases in the elution buffer, it displaces the His-tagged protein from the nickel ions, allowing for the selective recovery of the target protein. By controlling the concentration of imidazole, you can effectively wash away non-specifically bound proteins while retaining the His-tagged GFP. This selective binding and release mechanism makes imidazole vital for enhancing the specificity and efficiency of His-tagged protein purification processes.