For our larger protein constructs that contain fluorophores, adding a 6-residue histidine tag to either fluorophore tail may bind to nickel – using principles of affinity chromatography – will allow us to capture our proteins quickly and without disturbing native function. Ideally, we would only need this as one purification step after taking our cell lysate, centrifuging, and discarding the pellet. Histidine is an amino acid that will most readily associate with nickel, which is a transition metal. Nickel is immobilized on the chromatography column. Our protocol used here is adopted from Bornhorst et al. in the journal Methods of Enzymology.

Purification of Proteins Using Polyhistidine Affinity Tags Joshua A. Bornhorst and Joseph J. Falke. Principles and Techniques of Biochemistry and Molecular Biology (Seventh edition) By Keith Wilson and John Walker.