What is the purpose of washing the column in affinity chromatography?

Washing the column in affinity chromatography is essential for maintaining the purity and specificity of the proteins that are to be isolated, such as GFP in this context. During the binding phase, both the target protein (GFP) and various impurities or unbound proteins may attach to the column. The wash step allows for the removal of these non-specifically bound proteins and other contaminants that could interfere with the subsequent steps of purification.

This step is crucial because it ensures that only the target protein remains bound to the column. Once the unbound proteins and impurities are washed away, the purity of the target protein is enhanced, making it easier to subsequently elute and recover the GFP in a more concentrated and less contaminated form. Therefore, this washing process significantly improves the efficiency and outcome of the purification process.