![]() One of the most likely reasons that a his-tagged fusion protein does not bind to your selected immobilized metal affinity chromatography (IMAC) resin is that the tag itself is not accessible to coordinate with the metal, typically nickel or cobalt. However, a hidden tag or using a nonoptimized binding buffer can turn a straightforward purification into a nightmare. His-tagged protein purifications take advantage of histidine residues' affinity for transition metals such as cobalt (Co 2+) and nickel (Ni 2+). Where did things go wrong? Is there any way to salvage the purification? In this post, we discuss the most likely reasons a protein fusion didn't bind and options to overcome these complicationsįigure 1. After performing a trial purification and running the collected fractions on a gel, you find that the his-tagged protein flowed right through the resin. ![]() ![]() It took weeks to clone your his-tagged fusion protein and several more weeks to optimize the expression of your construct. ![]()
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