Subsequently, correlate the binding capacity (amount of beads used) with the amount of His-tagged target protein available. Some proteins naturally interact with immobilized metal ions and may bind to the resin. Typically, these ‘contaminating proteins’ interact with lower affinity than the His-tagged target and the His-tagged target is able to displace contaminating proteins from binding to the beads. If more beads are used, the His-tagged target protein can become the limiting reagent (not enough target to saturate the beads available binding capacity), and the impurities may remain bound to the beads and be carried throughout the purification.
Another option to reduce protein contaminant peaks is to employ proteolytic His-tag cleavage followed by reverse IMAC. First, an expression vector must be designed to contain a TEV site between the His-tag and the protein. Following purification with Ni-NTA Magnetic Beads, cleave the protein with TEV Protease (NEB #P8112), then load the sample onto the beads under similar or identical conditions to those employed for the initial purification. This will result in impurities re-binding to the beads while the untagged target protein is collected in the flow-through.