1. Different metal ions confer different binding affinity and specificity

His-tag purification is based on ionic interactions of a coupled metal ion and the Poly-His-tag. Therefore other metal ions can also fullfill the function of Nickel. Figure 1 shows the most frequently used metal ions for His-tag purification. Depending on the used metal ion the balance between the specificity and the affinity of the metal ion to the His-tag shifts. Nickel is a balanced choice between both extremes. Copper has the highest affinity, but Cobalt is the choice for the most pure His-tag purifications.

Fig. 1: Depending on which metal ion is coupled to NTA the balance between protein yield and purification specificity shifts. Nickel is a very balanced choice between the two extremes.