Why Did My Nickel Agarose Beads Lose Their Color?

by Simon Currie

Nickel agarose beads have a beautiful blue color. This blue color isn’t just for aesthetics; it is also a useful tracker of your purification or a way to tell if you’ve accidentally stripped the nickel ions off of your beads.

Ni²⁺ ions give nickel agarose beads their characteristic blue color. This blue color can fade or disappear completely when loading his-tagged proteins onto the column. Additionally, the color will completely fade to white if you’ve stripped the nickel ions off of the beads using EDTA or another metal chelator.

When loading his-tagged proteins onto your nickel agarose beads, it is quite common for their blue color to fade, or turn completely to white. In my experience, the stronger the color change, the more his-tagged protein there is bound to the column. If the color change is only due to protein being bound, then the blue color should return when you elute the his-tagged protein from your column (Figure 1).

nickel agarose beads losing color illustration

Figure 1. The column loses its blue color when EDTA strips nickel ions off of the agarose beads.

However, another reason your column could turn white is if you’ve stripped the nickel ions off of the beads with EDTA or another metal chelator. If this is why your column has turned white, then it will remain white even after you’ve tried to elute your protein. And you probably will have very little or no his-tagged protein in your elution as well.

The maximum compatible concentration of EDTA for different variations of nickel agarose beads are listed in Table 1. The higher the EDTA concentration you use, the lower the binding capacity of your nickel agarose beads will be. Additionally, if you’re reusing your nickel agarose beads and using high concentrations of EDTA, then you’ll need to strip, clean and recharge your beads more frequently.

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

In this article we go more in depth about the color of nickel agarose beads and why it can change. There are also links to many more educational resources and useful GoldBio products below. So, if you’re interested in learning more about nickel agarose beads or starting to purify his-tagged proteins, check them out!