How to Optimize Buffer Components for Nickel Agarose Beads

by Simon Currie, Ph.D.

Nickel agarose beads are compatible with a wide range of buffers. However, it is important to limit the amount of metal chelating agents, such as EDTA, and reducing agents, such as DTT and TCEP, in your buffers as they will impact the nickel beads purification performance and stability.

Nickel agarose beads are a really great tool for purifying his-tagged recombinant proteins. When leveraging the interaction between nickel ions and his-tags for affinity purification, it’s important to use protein purification buffers that are compatible with this interaction.

In this article, we’ll do a deep dive into buffer considerations when using nickel agarose beads, and help you maximize the yield of your his-tagged protein, as well as the stability of your beads so you can reuse them for multiple purifications.

Article Table of Contents

General protein purification buffer considerations

Salt

Imidazole

Denaturing agents

Buffer components to limit or avoid entirely

Metal chelators

Reducing agents

General protein purification buffer considerations

For GoldBio’s His-Tag Buffer Set and His-Tag Column Prep Protoco l, we recommend using 50 mM Phosphate Buffer pH 8, 300 mM NaCl, and 10 mM Imidazole as the binding buffer, and this is a great “baseline” buffer to use if you’re working with a new protein and unsure what is important to include in your purification buffers.

However, there are a pretty wide range of buffer components you can use for nickel agarose beads, and a few components to be cautious of, so let’s go through those one by one.

First off, let’s cover commonly used buffer components for his-tag purifications:

pH
salt
imidazole
denaturing agents

pH

It is recommended to keep the pH in the range of 5.5 to 8.5 for the stability of nickel agarose beads and to facilitate the interaction between nickel and the his-tag. The exact pH that you use can be dialed to fine-tune between higher protein yield, and better purity of your his-tagged protein.

His-tags bind to nickel ions through their histidine side chains, which has an ionizable imidazole group with an equilibrium dissociation constant (pK_a) of approximately 6.0 (Figure 1).

Figure 1. Ionization of histidine side chain (imidazole).

If you want more details about ionizable side chains and what exactly a pK_a is, this article is a great resource. But the takeaway point is that at pH values higher (more basic) than 6.0, histidine will be deprotonated (right side of Figure 1) and will bind more strongly to nickel ions. Conversely, at pH values lower (more acidic) than 6.0, histidine will be protonated (left side of Figure 1) and will bind weakly to the nickel ion.

How can you use this information to inform your his-tagged protein’s purification? Typically, researchers use basic pH (~ 7 to 8) to promote strong binding of their his-tagged protein to the nickel agarose bead. However, if you’re getting too many contaminating proteins in your elution, you could make the pH of your buffer more acidic (pH ~ 6 to 7) to disrupt weaker interactions between contaminating proteins and the nickel agarose beads.

Salt

You’ll want to include a salt such as sodium chloride in your buffer to prevent contaminating proteins from sticking to the nickel beads or to your protein of interest through spurious electrostatic interactions.

Typically, you’ll want to use somewhere between 150 and 500 mM NaCl in your binding and wash buffers. You can even go higher than 500 mM NaCl, as long as your protein is stable in that salt concentration, but you won’t want to go lower than 150 mM NaCl because your purity may decrease due to nonspecific protein binding.

Nickel agarose beads are also compatible with a wide range of salts, so while NaCl is most commonly used, exploring a different salt may be worthwhile if you’re still getting contaminating proteins in your elution, even in the presence of high concentrations of NaCl.

Imidazole

Imidazole is the side chain of histidine residues, so it is used to compete for binding to the nickel beads and releasing your bound his-tagged protein during the elution step (Figure 2).

While a higher concentration ( > 250 mM) is used for elution, using a lower concentration of imidazole (~10-50 mM) in the binding and wash buffers is very helpful for preventing contaminating proteins from binding to the nickel beads.

illustrates when high imidazole concentrations used in nickel beads

Figure 2. High concentrations ( > 250 mM) of imidazole (I) elute his-tagged proteins from nickel agarose beads.

Why do contaminating proteins bind to nickel beads in the absence of imidazole? Most proteins from the expression host will have at least one histidine residue, and several will have a few histidine residues in close proximity in their primary sequence that can simultaneously bind to a nickel ion (Hemdan et al, 1989; Salichs et al, 2009). The his-tag on your protein of interest, typically 6 to 10 histidine residues in a row, will bind more strongly to nickel, however. So, including ~ 10 – 50 mM imidazole in your binding and wash buffers will allow your his-tagged protein to bind while preventing contaminating proteins from binding to the nickel beads (Figure 3).

Low concentration of imidazole illustration for nickel agarose beads

Figure 3. Low concentrations ( ~ 10-50 mM) of imidazole (I) wash contaminating proteins off of nickel agarose beads, but not his-tagged proteins.

In summary, for the first three buffer components it’s good to include salt in your purification buffers, and then pH and imidazole concentrations can be dialed to toggle between enhanced purity and higher yield (Figure 4).

illustrates the relationship between imidazole and ph

Figure 4. There is a trade-off between enhanced purity and higher yield that can be toggled by adjusting the pH and the imidazole concentration in your binding and wash buffers.

Denaturing agents

One particular strength of nickel agarose beads, and their purification of his-tagged proteins, is compatibility with denaturing agents such as 8 M urea and 6 M guanidine hydrochloride. So, if your protein is expressing really strongly into inclusion bodies, then you can liberate your protein from their insoluble isolation using one of these denaturing agents, and purify your his-tagged protein while the denaturing agent is still present.

While there is no guarantee that refolding your protein of interest will be successful, if it does work, this purification route tends to have high yields and enhanced purity compared to purifying from the soluble fraction. (Currie et al, 2017a; Currie et al, 2017b).

Buffer components to limit or avoid entirely

Just because nickel agarose beads can withstand denaturing agents doesn’t mean they are compatible with all buffer components. In particular, it is important to limit nickel agarose bead exposure to two other buffer components: metal chelators and reducing agents.

Metal chelators

EDTA is a metal chelating agent, meaning that it binds to metals such as nickel. When included in your purification buffers, EDTA will strip nickel off of the agarose beads, which will limit your overall protein yield (Figure 5).

EDTA and nickel agarose beads illustration

Figure 5. EDTA strips nickel ions (blue circle) off of agarose beads.

EDTA is commonly used in protein purification buffers because it is an effective inhibitor of proteases. Host proteases are enzymes that will cut up your protein of interest. Adding EDTA to your buffer strips metal cofactors from these proteases, thereby inactivating them.

Since EDTA also strips nickel off of agarose beads, it is desirable, if possible, to eliminate or use as little EDTA as possible in your his-tag purification buffers.

Up to 1 mM EDTA can be used with most of GoldBio’s nickel beads, but the Highest Density Nickel Beads can withstand up to 20 mM EDTA (Table 1). So, if you need high EDTA concentrations in your purification buffers, then the Highest Density Nickel Beads are definitely the nickel beads that you should use.

Table 1. GoldBio Nickel Agarose Beads EDTA Compatibility

Nickel Agarose Bead Type	Maximum [EDTA]	GoldBio Catalog #
Highest Density Nickel	20 mM	H-390
Nickel NTA Magnetic	1 mM	H-351
Nickel NTA HTC	1 mM	H-355
Nickel NTA	1 mM	H-350
Nickel Agarose Beads (High Density)	1 mM	H-320
Nickel HTC	1 mM	R-202

Reducing agents

You also need to be aware of how much reducing agent to use in your purification buffers. Reducing agents such as b-mercaptoethanol (bME), dithiothreitol (DTT), and tris-(2-carboxyethyl)phosphine hydrochloride (TCEP HCl) are frequently used in protein purification buffers to keep proteins from forming aberrant disulfide bonds and aggregating.

However, at high concentrations these reducing agents will also reduce nickel ions from the Ni²⁺ state.

When this happens, the nickel column will turn from blue to brown, and the nickel ion will lose its capacity for binding his-tagged proteins.

Most GoldBio nickel agarose beads can withstand up to 5 mM DTT. However, the Highest Density Nickel Beads are compatible with up to 20 mM DTT (Table 2). So, if your protein is very redox sensitive and needs greater than 5 mM DTT in the purification buffers, then the Highest Density Nickel Beads are the clear choice for your purification needs.

Table 2. GoldBio Nickel Agarose Beads Reducing Agent Compatibility

Nickel Agarose Bead Type	Maximum [DTT]	GoldBio Catalog #
Highest Density Nickel	20 mM	H-390
Nickel NTA Magnetic	5 mM	H-351
Nickel NTA HTC	5 mM	H-355
Nickel NTA	5 mM	H-350
Nickel Agarose Beads (High Density)	5 mM	H-320
Nickel HTC	5 mM	R-202

Keep in mind that Tables 1 and 2 list the maximum compatible EDTA and DTT concentrations for each bead type. Limiting the concentration of these components in your purification buffers will enable you to reuse the nickel beads more times before they lose performance and need stripped, cleaned, and recharged.

However, if your protein needs higher concentrations of EDTA or DTT in the purification buffers, then you will need to frequently strip, clean, and recharge your nickel resin to keep it performing well.

Those are the key buffer components to keep in mind when designing purification buffers for your his-tagged proteins. However, this list is not comprehensive. If you have additional questions about buffer compatibility with GoldBio’s nickel agarose beads, see this resource.

Below we have links to many of GoldBio’s affordable and reliable products that will enable you to purify your his-tagged proteins, as well as some related resources to learn more and help troubleshoot – check them out!

References

Currie, S. L., Lau, D. K. W., Doane, J. J., Whitby, F. G., Okon, M., McIntosh, L. P., & Graves, B. J. (2017). Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5. Nucleic acids research, 45(5), 2223–2241. https://doi.org/10.1093/nar/gkx068

Currie, S. L., Doane, J. J., Evans, K. S., Bhachech, N., Madison, B. J., Lau, D. K. W., McIntosh, L. P., Skalicky, J. J., Clark, K. A., & Graves, B. J. (2017). ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain. Journal of molecular biology, 429(20), 2975–2995. https://doi.org/10.1016/j.jmb.2017.06.024

Hemdan, E. S., Zhao, Y. J., Sulkowski, E., & Porath, J. (1989). Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proceedings of the National Academy of Sciences of the United States of America, 86(6), 1811–1815. https://doi.org/10.1073/pnas.86.6.1811

Salichs, E., Ledda, A., Mularoni, L., Albà, M. M., & de la Luna, S. (2009). Genome-wide analysis of histidine repeats reveals their role in the localization of human proteins to the nuclear speckles compartment. PLoS genetics, 5(3), e1000397. https://doi.org/10.1371/journal.pgen.1000397