Why Did My Nickel Agarose Beads Turn Black or Brown?

by Simon Currie

Nickel agarose beads change from blue to a brown or black color when the nickel ions have been reduced from a Ni2+ to a Ni1+ or a Ni0 oxidation state. Reduction of nickel agarose beads most commonly occurs due to high concentrations of reducing agents such as DTT or TCEP in purification buffers. 

If you’ve purified his-tagged proteins with nickel agarose beads, then you know the characteristic brilliant blue color of these beads. When working with those beads, have you ever accidentally turned them a dark brown or black color?

Nickel agarose beads change from blue to a brown or black color when the nickel ions have been reduced from a Ni²⁺ to a Ni¹⁺ or a Ni⁰ oxidation state. Reduction of nickel agarose beads most commonly occurs due to high concentrations of reducing agents such as DTT or TCEP in purification buffers. 

The maximum concentration of DTT recommended for various types of GoldBio’s nickel agarose resins listed in Table 1. TCEP is more compatible with nickel beads, so you can use at least that same concentration for TCEP, though I wouldn’t recommend going much higher.

Table 1. GoldBio Nickel Agarose Beads DTT Compatibility

So, if your nickel agarose beads changed to dark brown or black (Figure 1), you may want to use a fresh batch of nickel beads, or strip, clean, and recharge the beads you have with fresh Ni²⁺ ions, and start your purification again with lower concentrations of reducing agents in your purification buffers.

Figure 1. DTT reduces Ni²⁺ ions on the agarose beads, changing the column color from blue to dark brown or black.

Note that if you’re using high concentrations of DTT or TCEP that are close the maximum compatible concentration, then you may still see some slight discoloration of the beads. You can proceed with your purification as long as the reducing agent concentration is lower than the maximum amount. However, the lower the concentration of reducing agents are in your purification buffer, the less discoloration and the higher the binding capacity will be for your beads. So, decrease that reducing agent concentration as much as possible in your buffers while still keeping your protein of interest stable.

If you want to learn more about the color of nickel agarose beads, and why it changes during purifications, check out this article. And we also have more tips for purifying his-tagged proteins, in the related resources section below. And if you’re ready to purify some proteins with nickel agarose beads in the lab, check out GoldBio’s reliable resources that can aid your research at a competitive price.

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nickel agarose beads Nickel Beads nickel ida nickel nta nickel resin protein purification Simon Currie

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