His-Tag Metal Affinity Cations: What’s the difference again?

Unless you’ve done the equivalent of a post-doc on his-tag protein purification, you have probably always wondered about the various metal cations available and what each one does for your purification needs. Today, we get to look into the sometimes-mysterious world of ion affinity!

Immobilized-Metal Affinity Column (IMAC) was first described in 1975 by Porath et al. and, in the decades since, has become one of the most popular and widely used chromatography techniques today. Porath used iminodiacetic acid (IDA) as a chelating ligand to fix a metal cation to agarose beads (still the technique used in many commercially available affinity resin). In the meantime, scientists began genetically modifying recombinant protein to include an oligohistidine extended (His-tagged) polypeptide sequence. This His-tag tail made it easier for proteins to bind to Porath's affinity resin. A second chelating ligand, nitrilotriacetic acid (NTA), was later developed in 1987. NTA coordinates with the divalent metal ion with four valencies (tetradentate), leaving two valencies for interaction with the imidazole rings of histidine residues. IDA, on the other hand is tridentate, coordinating its divalent metal ions with three valencies, leaving another three for histidine association. Though there are some specific differences between the use of the two ligands, protein recovery between IDA cross-linked resin and NTA cross-linked resin are usually fairly similar.

The choice of metal ions is often highly dependent on the application. Trivalent cations, such as Al³⁺, Ga³⁺, or Fe³⁺, or even tetravalent ions, like Zr⁴⁺, are preferred for binding of phosphoproteins or phosphopeptides. But divalent cations, such as Cu²⁺, Ni²⁺, Zn²⁺ and Co²⁺ are most often used for purification of His-tagged proteins.

At Gold Bio, you will find all four of these divalent cations cross-linked to agarose beads for your his-tag purification needs. They are additionally described as either a High Density or Low Density resin. Below is a graph describing the characteristics of the cations and their general strength/weakness for protein affinity and specificity.

Affinity can best be described as the strength of the cation’s binding capacity. A cation with high affinity will bind more strongly to his-tagged protein than one with less affinity. Specificity is best described as the selectivity of the cation’s binding capacity. A higher specificity means that the cation will bind to a narrower group of protein. And of course, these two traits are at odds with each other. As seen above, Cu²⁺ has a high affinity, but low specificity. Alternatively, Co²⁺ has a high specificity, but a low affinity. So if you aren’t concerned with protein purity and want to bind as much protein as possible, you will likely want to use Copper. But if you have a fairly diverse protein sample and want to purify a very particular protein from the mix, you will likely want to use Cobalt.

Ni²⁺and Zn²⁺ cations fall somewhere in between, often giving you the best of both worlds. Nickel has a fairly good affinity, with a slightly low specificity, whereas Zinc has a fairly good specificity with a slightly low affinity. Which one you pick should depend completely on what you want to get out of your purification. Nickel seems to be the most widely used cation, with a good affinity efficiency. But its nonspecificity to any endogenous proteins containing histidine clusters often makes it a frustrating choice. In those cases, a better choice might be a Zinc resin, giving you comparable affinity but a much better specificity overall.

The High Density and Low Density resins often work to “bump” the cation’s association. A High Density resin helps to increase the affinity of the resin, while a Low Density resin helps to increase specificity. Both Ni²⁺ and Zn²⁺ are available in both High and Low Density resins (for your specific needs), but the Cu²⁺ is only available in Low Density and Co²⁺ is only available in High Density (for obvious reasons).

We hope this helped and if you still have any questions, please contact us at [email protected]. Happy purifying!

Block, Helena, et al. "Immobilized-metal affinity chromatography (IMAC): a review." Methods Enzymol 463 (2009): 439-473.

Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975). Metal chelate affinity chroma-
tography, a new approach to protein fractionation. Nature 258, 598–599.

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