Why Are Nickel Agarose Beads Blue?

by Simon Currie

The characteristic blue color of nickel agarose beads comes from the 2+ oxidation state of the nickel ions. Color is also a useful indicator for your beads as it often fades when his-tagged proteins bind to the column, and the color will change drastically if nickel is reduced or stripped from the beads.

Many experimental procedures in science have no way to monitor in real time how the procedure is going. Think about it, we’re usually pipetting colorless liquids from one tube to another, and then we have to wait hours or even days to evaluate whether we did it correctly.

In contrast to the many achromatic experiments, using nickel agarose beads to purify his-tagged proteins satisfyingly has a color indicator that can let you know if you’re on the right track.

The characteristic blue color of nickel agarose beads comes from the 2+ oxidation state of the nickel ions. Color is also a useful indicator for your beads as it often fades when his-tagged proteins bind to the column, and the color will change drastically if nickel is reduced or stripped from the beads.

In this article:

Nickel color and oxidation state

Blue fading when his-tagged proteins bind to the beads

Column turns white when nickel ions are stripped off of the beads

Column turns black or brown because nickel ions are reduced

Nickel agarose beads are normally a brilliant light blue. However, their color can change during purification or cleaning steps, and this color change is a convenient indicator of how your beads are doing.

The three most common color changes for nickel agarose beads are (Figure 1):

• Fading or complete loss of blue color as his-tagged protein binds to the column.
• Complete loss of blue color due to stripping nickel from the agarose beads.
• Beads turn black or brown due to reduction of Ni²⁺ ions.

Figure 1. Blue nickel columns (left) can have A) their color fade as protein binds, B) completely lose their color if nickel is stripped off the beads, or C) turn black or brown if the nickel ions are reduced.

Nickel color and oxidation state

Nickel ions on agarose beads normally have a +2 oxidation state. This +2 oxidation state gives the nickel agarose beads their characteristic blue color, and it also enables nickel to bind to his-tagged proteins.

As a quick review of oxidation and reduction, you may have heard the mnemonic phrase “LEO the lion says GER”. This phrase reminds us that losing electrons is oxidation, and gaining electrons is reduction.

Nickel being in a +2 oxidation state means that it has lost two electrons, and it will be energetically favorable for it to share two electrons with its binding partners. That’s where the his-tag comes in. The imidazole side chain of histidine will readily share one of its electron lone pairs with nickel. And by usually having at least six histidine residues in a row in the his-tag, there are two imidazole groups in close proximity ready to donate electrons to Ni²⁺ (Figure 2).

Figure 2. Imidazole side chains from the his-tag share an electron lone pair to bind with Ni²⁺ ions (molecules not drawn to scale).

When the color of the nickel beads changes, it really means one of two things: either the oxidation state of nickel has changed, or the nickel ions are no longer on the resin.

As we discuss below, when the beads turn brown or black, this means nickel has been reduced. Since nickel has gained electrons, it will now have a weaker interaction with his-tags because it doesn’t need to share imidazole’s lone pair anymore. So, that’s the coordination chemistry basis of nickel’s color, and why it matters for binding his-tagged proteins.

Blue fading when his-tagged proteins bind to the beads

Nickel agarose beads fading into a lighter blue or whitish color could be a good sign if you’re in the middle of a purification. As your his-tagged protein binds onto the nickel column, its color should fade into a lighter blue. When my column would look really light, like a whitish color, I got excited because that usually meant that I would have a lot of his-tagged protein bound to the column. 

If this is the reason why your nickel column is turning lighter blue or white, then when you elute your his-tagged protein off of the column, the brilliant blue color should mostly return to the beads (Figure 3). However, if the blue color doesn’t return after elution, that could mean that you inadvertently stripped the nickel ions off of the agarose beads during your purification. We’ll discuss that scenario in the next section.

Figure 3. The blue color of the column may fade or disappear when his-tagged proteins are bound, but will return when they’re eluted.

Column turns white when nickel ions are stripped off of the beads

Another reason that your column would turn white is if you’ve stripped the nickel ions off of the agarose beads. Metal chelators, such as EDTA, bind to the nickel ions and will strip them off of the beads (Figure 4).

Figure 4. When EDTA strips nickel ions off of the agarose beads, the column loses its blue color.

For this reason, it’s important to minimize the amount of EDTA in your purification buffers, or avoid it altogether. The maximum amount of EDTA for different variations of GoldBio’s nickel agarose resins are listed in Table 1.

Table 1. GoldBio Nickel Agarose Beads EDTA Compatibility

The Highest Density Nickel beads are compatible with up to 20 mM EDTA, so if you have to use a lot of EDTA in your purification buffers, then those are the beads you should use.

Whichever beads you use, it’s true that the more EDTA you have in your buffers, the more nickel ions will be stripped each purification. Over time, if your nickel beads are losing their brilliant blue color, that is a sign that you should strip, clean, and recharge them with fresh nickel ions to restore their blue color and, more importantly, their binding capacity for his-tagged proteins.

Column turns black or brown because nickel ions are reduced

If your nickel column turns a brown or black color it means that the nickel ions have been reduced from their Ni²⁺ state to Ni¹⁺ or Ni⁰ (Figure 5). Nickel will no longer bind his-tagged proteins in this state (or bind them very poorly). So, if you see a strong color change to brown or black, you should not use these beads for protein purification.

Figure 5. DTT reduces Ni²⁺ ions on the agarose beads, changing the column color to dark brown or black.

Nickel is reduced when it comes into contact with a reducing agent. In the context of protein purification buffers, this reducing agent is usually DTT or TCEP. Table 2 lists the maximum concentration of DTT compatible with different versions of our nickel agarose beads. TCEP is more compatible with nickel beads, so you can use at least the listed concentration, though I don’t recommend going much higher.

Table 2. GoldBio Nickel Agarose Beads DTT Compatibility

If your beads turn black or brown, not all is lost. With a simple cleaning and recharging protocol, you can have these beads working well again. We describe this protocol in more detail in this article. But in brief, you’ll strip the reduced nickel off of the beads with a high concentration of EDTA, clean the beads if you want, and then add back fresh blue Ni²⁺ ions. Your beads are now good to go again. Just remember to limit the reducing agent this time!

That being said, it doesn’t always make sense to use your precious time regenerating nickel agarose beads. GoldBio offers excellent pricing on beads, so it may be worth it to save your time and quickly start over with fresh beads.

If you have relatively high concentrations of DTT or EDTA in your buffer, it is normal to see some slight discoloration of your nickel beads. If these components are absolutely required at the concentrations that you are using, then go ahead and proceed with your purification as long as the concentrations are within the limits listed in Tables 1 and 2. Limiting, or eliminating, these components in your purification buffers will minimize discoloration and maximize his-tagged proteins binding to the nickel beads.  

If you’re ready to purify his-tagged proteins with nickel agarose beads, check out GoldBio’s high-quality products at an affordable price below. In particular, our Highest Density Nickel beads are compatible with higher concentrations of DTT and EDTA, so those may be just what you need if you’ve been turning your beads brown or white!

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his-tag nickel agarose beads nickel resin protein purification Simon Currie

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