Nickel Affinity Chromatography - The Complete Overview

by Simon Currie

Nickel affinity chromatography is a technique used to purify proteins that contain a polyhistidine tag (his-tag). Nickel chromatography is a popular choice for affinity purifications due to its rapid and specific binding, speed, flexible and gentle elution conditions, and affordability.

Do you have a favorite restaurant that you love because you know exactly how great the experience is going to be? There are probably a few restaurants out there with slightly better food, service, or pricing. But your favorite one is tough to beat because it’s great in all of those categories.

Nickel chromatography is like that in terms of affinity protein purification techniques. Sure, other techniques might be a little bit better at binding specificity or better in certain circumstances, but nickel chromatography is a go-to technique because it works reliably for a wide-range of proteins.

In this article we’ll discuss nickel agarose beads, the general principles of how nickel chromatography works, and compare nickel chromatography with other affinity purification techniques.

In this article:

Nickel agarose beads

Nickel affinity chromatography

Nickel bead binding capacity and buffer compatibility

What the color of the nickel beads tells me about my purification

Comparing with other affinity chromatography techniques

Nickel agarose beads

Nickel is conjugated to agarose beads using one of two chelating agents: iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) (Figure 1).

Figure 1. NTA (right) makes one more bond with Ni²⁺ than IDA (left) which impacts protein binding yield and specificity.

GoldBio sells both forms of nickel beads, NTA and IDA, and overall, they are both very similar in terms of how they perform with nickel affinity chromatography. The key difference is that nickel NTA typically is a little bit better in terms of protein purity.

However, IDA resin can give slightly higher protein yield and is a little more affordable. Nickel NTA and IDA are very interchangeable, but you can check out this article for more details on the slight differences between them.

The other thing to point out is that nickel affinity chromatography is the most commonly-used form of immobilized metal affinity chromatography (IMAC). Other metals can be used instead, such as cobalt. Cobalt also binds to his-tagged proteins, typically with higher specificity which means less contaminating proteins compared to nickel.

Nickel affinity chromatography

The nickel on agarose beads binds to proteins through polyhistidine tags, known as his-tags, that usually range from 6 to 10 histidine residues long (Figure 2).

Figure 2. His-tagged proteins (green) bind to nickel agarose beads.

After binding his-tags to the nickel beads, the beads are washed, then eluted by raising the concentration of imidazole in the elution buffer. Imidazole looks just like the side chain of histidine residues, so excess concentrations of imidazole, usually 250 to 500 mM imidazole, are used to elute his-tagged proteins from the nickel agarose beads (Figure 3).

Imidazole elutes his-tagged proteins from nickel bead.

Figure 3. Imidazole (purple) elutes his-tagged proteins from the nickel bead.

The longer the his-tag, the tighter it will bind to nickel. This means you can usually obtain more pure protein with a longer his-tag by performing a more stringent wash step (higher imidazole concentration). To learn more about his-tag length and how it impacts purification, see this article.

Nickel bead binding capacity and buffer compatibility

One of the first questions you’ll ask yourself when using nickel beads to purify his-tagged proteins, or for other experiments, is how much should I use? To figure out the approximate volume of nickel agarose beads to use, you’ll need to know the binding capacity of your beads. The binding capacity is how much his-tagged protein a particular type of nickel agarose beads can bind. For GoldBio’s nickel agarose beads the binding capacity ranges from 6 – 80 mg/mL (Table 1).

Table 1. GoldBio Nickel Agarose Bead Binding Capacity

Nickel Agarose Bead Type	Binding Capacity	GoldBio Catalog #
Highest Density Nickel	80 mg/mL	H-390
Nickel NTA Magnetic	75 mg/mL	H-351
Nickel NTA HTC	60 mg/mL	H-355
Nickel NTA	50 mg/mL	H-350
Nickel Agarose Beads (High Density)	25 mg/mL	H-320
Nickel HTC	6 mg/mL	R-202

For a binding capacity of 80 mg/mL, this means that 80 mg of protein should bind to 1 mL of agarose resin. You should view that number as an idealized upper limit, which will likely be a little bit lower in reality depending on parameters such as the size of your his-tagged protein and the exact purification buffers that you’re using.

We cover those details and more in this article about nickel agarose bead binding capacity.

In terms of the buffers that you’re using, there are a couple of key components that you need to be careful with when using nickel agarose beads: metal chelators and reducing agents such as EDTA and DTT (Table 2).

Table 2. GoldBio Nickel Agarose EDTA and DTT Maximum Concentrations

Nickel Agarose Bead Type	Maximum [EDTA]	Maximum [DTT]
Highest Density Nickel	20 mM	20 mM
Nickel NTA Magnetic	1 mM	5 mM
Nickel NTA HTC	1 mM	5 mM
Nickel NTA	1 mM	5 mM
Nickel Agarose Beads (High Density)	1 mM	5 mM
Nickel HTC	1 mM	5 mM

As we will discuss in the next section, using too much EDTA or DTT will strip the nickel off of your agarose beads, or render them incapable for binding to his-tags.

What the color of the nickel beads tells me about my purification

Biochemistry often involves pipetting colorless liquids from tube to tube, and you have no idea if anything is working until days, weeks, or even months later. In that context, working with nickel agarose beads is really satisfying in that the beads themselves serve as a real-time color indicator of how your purification is going.

Nickel agarose beads themselves are a brilliant blue (Figure 4). As you’re doing your purification, it is normal for the blue color to fade or turn completely to white when his-tagged proteins are bound to the column. However, the blue color should at least partially return when you elute your protein off of the column.

Figure 4. Nickel beads are blue (left), but that color can change during protein purification.

If the blue color doesn’t return and your column is still white after elution, this means that you stripped the nickel ions off of the agarose beads, likely by using too much EDTA in your buffers.

Alternatively, if your column turns dark brown or black during the purification, this means that you’ve reduced the nickel ion on the agarose beads, likely due to too much reducing agent such as DTT or TCEP in your purification buffers. The reduced form of nickel binds very poorly to his-tagged proteins, so if you see this color change, you’ll likely want to stop your purification.

For either of these cases, you may want to try purifying your protein with less EDTA or reducing agent in your buffers. GoldBio’s Highest Density Nickel Beads are compatible with up to 20 mM of EDTA or DTT (Table 2), so if you cannot reduce these components in your buffer then that is probably the best form of nickel beads for your experiment. If your protein is unstable in less than 20 mM EDTA or DTT, then you could also try using a different purification tag, such as those discussed in the next section.

If either of these situations occurs, you can always strip, clean, and recharge your nickel agarose beads as described in this article.

Comparing with other affinity chromatography techniques

His-tags are probably the most frequently used affinity purification tags, but they’re certainly not the only ones. Other examples of affinity tags include FLAG-tags, strep-tags, and GST-tags which bind to antibody-, streptavidin-, and glutathione-conjugated agarose beads, respectively.

If you want to learn more about FLAG- and strep-tags and how they work, check out this article. We also cover GST-tags in a lot more detail in this article.

So why are his-tags and nickel agarose beads so frequently used? Just like your favorite restaurant, they aren’t necessarily the best at everything, but they’re pretty darn good in most areas, which makes them a versatile purification and detection tool.

Impact of tag

His-tags are generally pretty innocuous, meaning they usually do not change a protein’s structure, function, or oligomerization state. In contrast, FLAG-tags are very positively charged and strep-tags are quite hydrophobic. The more extreme nature of these other tags means that they will sometimes have more impact on a protein compared to his-tags.

Solubility tags such as GST are quite a bit bigger than smaller affinity tags such as his-tags. This means that GST sometimes dominates the properties of fusion proteins and can more frequently impact a protein’s function. Additionally, GST dimerizes with itself and so GST-fusion proteins also often dimerize even if your protein of interest is normally a monomer (Singh et al, 1987). This dimerization can also impact the fusion protein’s function (Tudyka and Skerra, 1997).

Cost of Reagents

When considering the cost of purification, the main purchases are going to be the affinity agarose beads and the component that you’re using for elution. For nickel affinity purification, this is nickel agarose beads and imidazole for eluting. Compared to the other tags we mentioned above, these reagents are less expensive which is a major reason that nickel affinity chromatography is so widely used (Table 3).

Table 3. Reagents and relative costs for different types of affinity chromatography.

Affinity Tag	Beads	Eluent	Relative Cost
His-Tag	Nickel	Imidazole	$
GST-Tag	Glutathione	Glutathione	$$
FLAG-Tag	aFLAG Antibodies	FLAG Peptide	$$$
Strep-Tag	Streptavidin	Biotin	$$$$

Also, as we discussed above, nickel agarose beads are easily stripped, cleaned, and recharged, making these beads even more affordable if you choose to follow this protocol.

Elution Conditions

You elute your his-tagged proteins off of nickel agarose beads with imidazole. Imidazole is well-tolerated by most proteins, though there are rare cases of proteins that precipitate in high imidazole solutions.

Elution conditions for other affinity tags are also generally well tolerated by proteins, so this really isn’t a reason to try one tag or another first. However, if you notice that your protein is aggregating after elution with any of these affinity tags, then it may be worth trying a different tag to avoid that particular eluent.

Binding specificity

His-tags have lower binding specificity compared to strep-, FLAG-, and GST-tags, which is the major limitation of nickel affinity chromatography (Figure 5) (Kimple et al, 2015). So, if you’re only doing a one-step affinity purification and purity is key, then you probably will want to use one of these other tags.

Figure 5. Affinity tags ordered from most to least specific binding: strep-, FLAG-, GST-, and his-tags.

However, nickel affinity purifications are frequently used as a first step in an overall purification protocol that might also include ion-exchange, hydrophobic-interaction, or size-exclusion steps. If that is the case for your purification, then the other strengths of his-tags may outweigh this limitation of relatively poor binding specificity.

So, that’s an overview about nickel affinity chromatography and why it is such a popular choice for protein purification. We’ve linked to many articles that delve deeper into subtopics that we briefly touch on here, so look into those below and throughout this article. Additionally, if you’re ready to start purifying his-tagged proteins, GoldBio has lots of affordable and reliable reagents to aid your research so check out those options as well.

References

Kimple, M. E., Brill, A. L., & Pasker, R. L. (2013). Overview of affinity tags for protein purification. Current protocols in protein science, 73, 9.9.1–9.9.23. https://doi.org/10.1002/0471140864.ps0909s73

Singh, S. V., Leal, T., Ansari, G. A., & Awasthi, Y. C. (1987). Purification and characterization of glutathione S-transferases of human kidney. The Biochemical journal, 246(1), 179–186. https://doi.org/10.1042/bj2460179

Tudyka, T., & Skerra, A. (1997). Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli. Protein science : a publication of the Protein Society, 6(10), 2180–2187. https://doi.org/10.1002/pro.5560061012

Nickel Affinity Chromatography - The Complete Overview

Nickel agarose beads

Nickel affinity chromatography

Nickel bead binding capacity and buffer compatibility

What the color of the nickel beads tells me about my purification