At which step is the supernatant first introduced into the chromatography column?

The introduction of the supernatant into the chromatography column is a crucial step in the purification process, specifically occurring during the step where the clarified mixture containing the target protein (in this case, Green Fluorescent Protein or GFP) is loaded onto the column.

In the purification protocol, prior to this step, the cells are typically lysed to release the proteins, and then the mixture is centrifuged to separate the soluble proteins from cell debris. The supernatant, which contains the soluble proteins, is then collected. Step 8 is designated as the point where this supernatant is applied to the chromatography column. This is when the target protein can begin to interact with the resin, allowing for separation based on specific properties of the protein, such as size or affinity.

The importance of this step can't be understated; the successful binding of GFP to the column is pivotal for subsequent purification steps, which may include washing and elution to isolate the protein further. Loading at the appropriate moment ensures the resin is conditioned properly and that the maximum amount of target protein is retained in the column for effective purification.