How much equilibration buffer should be added to the column during the preparation step?

The correct amount of equilibration buffer to use during the preparation step is 2 mL. This volume is typically sufficient to ensure that the column is properly equilibrated before loading the sample. Equilibration is a critical step that prepares the column by stabilizing the conditions, such as pH and ionic strength, which are necessary for optimal binding of the target protein—in this case, green fluorescent protein (GFP).

Using 2 mL allows for adequate saturation of the resin within the column, ensuring that when the sample is introduced, the binding interactions can occur effectively. If too little buffer is used, the column may not reach the desired conditions for binding, which could lead to reduced yield and purity of the protein. However, using excessive buffer can lead to unnecessary waste and potentially dilute the sample, affecting the overall efficiency of the purification process. Thus, 2 mL strikes a balance that is often recommended in standard protocols for protein purification.