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What’s the Difference Between Nickel NTA and Nickel IDA Agarose Beads?

by Simon Currie, Ph.D.

While very similar overall, the fundamental difference between NTA and IDA is that NTA forms 4 bonds with a nickel ion whereas IDA makes 3 bonds. Slight differences in purification performance and pricing influence whether Ni NTA or Ni IDA agarose beads will best suit your needs.

There are a lot of options when choosing nickel agarose beads for purifying your his-tagged proteins. So, the decision on which beads to use can easily get overwhelming.

One relatively simple part of that decision is the linker that conjugates the nickel ion to the agarose beads: nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA). NTA and IDA are chelating agents that conjugate nickel ions to agarose beads for the purification of his-tagged proteins.

In this article, we’ll dive a little deeper about the similarities and differences between NTA and IDA in how they’re used for protein purification.

Article Table of Contents:

NTA and IDA chelating nickel ions

Choosing between NTA and IDA

Related Resources

NTA and IDA chelating nickel ions

NTA and IDA both chelate, or bind to, nickel ions and other similar transition metals like cobalt. However, NTA and IDA engage nickel ions a little differently.

First, we need to cover a few basics of inorganic chemistry for context on why NTA and IDA bind differently. In coordination chemistry terms, the nickel ion is the central metal atom, and the surrounding molecules (NTA, IDA, and protein his-tags in this context) are called ligands.

A nickel ion with a +2 charge (Ni²⁺), the most common form of nickel, can engage up to 6 bonds with other molecules (Figure 1).

Figure 1. Illustration of Nickel 2+ ions (blue) can make 6 bonds (gray) to other molecules.

NTA binds to 4 of the 6 available sites on nickel, leaving the other two sites available for interaction with his-tagged proteins.

IDA only binds to 3 of the available sites on nickel, leaving the other three sites available for interaction with his-tagged proteins (Figure 2).

IDA vs. NTA beads and binding illustration

Figure 2. IDA makes three bonds with nickel ions (left) whereas NTA engages four bonds (right).

NTA and IDA similarities and differences

Both NTA and IDA chelate nickel ions, which engage his-tags (Figure 3). So, both types of nickel agarose beads are used for purifying his-tagged proteins.

Figure 3. Agarose beads (gray) use IDA or NTA (pink) to chelate nickel ions (blue) and capture his-tagged proteins (green).

From a “big-picture” perspective, NTA and IDA nickel beads are really interchangeable in that regard. However, there are some slight differences that are worth considering when deciding between Ni NTA and IDA agarose beads.

NTA forms an additional bond with nickel ions, leaving Ni²⁺ with one less site to bind to proteins. This means that IDA beads can give a slightly higher yield of the his-tagged protein. However, this additional binding site on nickel frequently means that purifications with IDA beads tend to have a few more impurities compared to Ni NTA beads (Figure 4).

Figure 4. Hypothetical SDS PAGE gel of a purification with either nickel IDA or NTA beads. In general, the IDA beads will have a higher yield for your his-tagged protein of interest but may also have more contaminants (less purity).

The additional bond also means that nickel ions stay on the resin better with Ni NTA beads, whereas it leaches off of the Ni IDA beads over time. This means that if you plan on reusing your beads for additional purifications, that Ni IDA beads will need stripped, cleaned, and recharged more frequently than Ni NTA ones.

For an extreme analogy, imagine riding a pretty wild rollercoaster. It’s super fast, has a ton drops and goes forward and backwards abruptly (again, imaginary here). It would be like having one less strap holding you in. But our NTA rollercoaster would have an extra strap – you would probably be glad to have that extra strap when you are hundreds of feet up in the air hanging upside down, right?

The last comparison to keep in mind is that Nickel IDA beads tend to be a little bit more affordable than Nickel NTA beads.

So, to reiterate their differences, Ni IDA beads are more affordable and have a higher protein purification yield whereas Ni NTA beads keep the nickel ion more stably bound to the resin and often have better purity.

Choosing between NTA and IDA

So, which one should you use for purifying his-tagged proteins - Ni NTA or IDA agarose beads? In the majority of scenarios, their performance will be similar enough that it is not really a big difference which one you choose. And in those cases, you may as well go with the Ni IDA beads to save a little money.

But there are definitely times when it will make more sense to go with Ni NTA beads for your purification.

One reason is if you plan to reuse your nickel agarose beads multiple times. Ni NTA beads more stably retain their nickel ions, so you can reuse them more times before you need to strip and recharge them.

Another reason to use Ni NTA beads is if enhanced purity of your protein sample is crucial and you’re not going to do any additional downstream purification steps. Often, affinity purification is only the first step in an overall purification scheme that may include ion exchange or size exclusion chromatography. If you’re doing either of those steps after your nickel purification, then you may separate out any additional contaminating proteins that bind to Ni IDA beads anyway. But if you’re not doing any extra purification, then Ni NTA beads are your best shot at having a pure protein sample after only one affinity purification step.

So, now you know the similarities and differences between Ni IDA and Ni NTA beads. If you’re still unsure on which one to get, you could always buy both and compare them head-to-head, but I’d say this is only really necessary if you’re going to be doing a ton of purifications and want to see which one you should go with over the long haul.