How should supernatant be added to the column during loading?

When loading supernatant onto a purification column, allowing it to drip down the side of the column is the most appropriate method. This technique ensures a gentle and even distribution of the sample across the resin in the column. By dripping it down the side, you minimize the risk of disturbing the packing material inside the column, which can lead to uneven flow patterns and impaired separation of biomolecules. This method also helps to maintain the integrity of the column and provides better contact between the supernatant and the stationary phase, enhancing the adsorption of the target protein, like GFP, to the resin.

Pouring from a height or directly into the drainage hole might create turbulence, which can negatively affect the separation process and lead to poor results. Additionally, mixing while adding could introduce air bubbles or cause shearing of sensitive molecules, compromising the sample and the purification process. Therefore, allowing the supernatant to gently drip down the side of the column facilitates optimal loading conditions and contributes to the overall success of the purification procedure.