Stripping and recharging Nickel Beads Thumbnail

How to Strip, Clean, and Recharge Nickel Agarose Beads

by Simon Currie, Ph.D.

Like other resins, preparing nickel agarose beads for reuse involves cleaning with solutions that contain high salt, basic pH, or organic solvents. However, these beads are different in that the nickel ions need to be stripped off of the resin before cleaning, and added back on afterwards.

Have you ever heard the adage “reduce, reuse, recycle?” When it comes to purifying proteins with agarose beads, we certainly hope you aren’t reducing that; the world needs more research. But you may want to consider reusing your agarose beads to save precious research dollars that can be put towards other vital experiments. If you’re wondering how to reuse your nickel agarose beads, the great news is that it’s really easy to clean them up and prepare them for another round of purification.

Nickel agarose beads are used to purify his-tagged proteins. Cleaning and regenerating agarose beads for protein affinity purification is a great way to keep your resin in optimal form and reduce the cost of buying new beads every time. In this article, we’ll discuss how to strip, clean, and recharge nickel resin so you can successfully purify his-tagged proteins over and over again.

When to regenerate nickel agarose resin?

Nickel agarose beads are used to purify his-tagged proteins. After you’ve finished your purification, you may want to regenerate your nickel agarose beads.

Regenerating refers to the entire process of stripping the old nickel ions off of the resin, cleaning the beads, and then adding new nickel ions back onto the beads.

If you are going to purify the same exact protein again in the future, then you can simply rinse your elution buffer out with a few column volumes of deionized water (dIH₂O), and then store the beads in 20% ethanol until you’re ready to use them again. Unless you’re using high concentrations of EDTA or reducing agents in your buffers, nickel agarose beads should perform well for several purifications and can be reused with just this rinsing step.

However, if one of the following situations applies to you, then you will want to strip, clean, and regenerate your nickel agarose beads to get the best performance possible.

You will reuse the nickel agarose beads to purify a new protein
You used high concentrations of EDTA or reducing agents in your buffer that are rapidly deteriorating the nickel beads
You’ve reused your beads several times already, and want to restore the binding capacity to near its original levels

Any of these scenarios are good reasons to strip, clean, and recharge your nickel agarose beads to maintain their peak performance.

Buffers for stripping, cleaning, and recharging nickel agarose beads

To prepare your beads for regeneration, pipette them into a column. For each of these steps you’ll simply pipet the recommended solution into the column and catch the flow-through in a beaker sitting below the column (Figure 1).

Figure 1. Pour your used nickel agarose beads in a column and pipet the stripping, cleaning, and recharging solutions into the column, collecting the flow-through in a beaker below.

There are distinct stripping, cleaning, and recharging steps for regenerating nickel agarose beads. Let’s take those sections one-by-one, and focus on which buffers you’ll need for each step.

Nickel stripping buffer

First, we need to get the nickel ions off of the resin. This is because nickel will react with some of the harsh reagents in the cleaning step, and ruin the beads.

The nickel stripping buffer we recommend in our official protocol is:

20 mM sodium phosphate pH 7.0, 500 mM NaCl, 50 mM EDTA

EDTA is really the “active” ingredient here, which will bind to the nickel ions and strip them off of the resin (Figure 2). During our actual purifications, we want to limit EDTA’s concentration to avoid stripping the nickel off of the column, so we typically use less than 1 mM.

The Highest Density Nickel beads are the exception to this rule. They are specifically designed to withstand up to 20 mM EDTA.

Now that we are done with our purification and intentionally stripping the nickel ions off of the column, however, it is useful to use 50 mM EDTA to get all of the nickel out of there.

Figure 2. High concentrations of EDTA strip nickel ions off of agarose beads.

500 mM NaCl helps clean any cellular debris that is sticking to the agarose beads through electrostatic interactions, so it’s nice to have in there too. The pH of the buffer really isn’t too critical here, just use something in a normal pH range that is compatible with the resin (between pH 3 - 12).

Before adding the stripping solution, go ahead and rinse your column out with 2 column volumes (CVs) of dIH₂O. This is a prudent step that I like to perform before and after adding all of the solutions in this article, and we’ll discuss in the next section why that is.

Then, add 10 CVs of the nickel stripping solution, followed by another 2 CVs of dIH₂O.

Resin cleaning buffers

Now that the nickel ions are off of the resin, it’s time to give those beads a nice thorough cleaning. The most commonly used cleaning solutions are:

1.5 M NaCl
1 M NaOH
30% isopropanol
70% ethanol
0.5% non-ionic detergent in 0.1 M acetic acid

These are five distinct cleaning solutions, so you wouldn’t combine all of the above components into a single cleaning buffer. Also, you don’t have to use all of these cleaning solutions. If you use more than one of them, make sure to rinse your column with several CVs of dIH₂O before and after using each cleaning solution.

It’s important to do those intermediate dIH₂O rinses because some of these solutions are incompatible with one another. 70% ethanol, for example, will precipitate out the salt in 1.5 M NaCl, and that precipitation will clog your beads. By rinsing with dIH₂O in between washes you avoid any of these potential incompatibilities.

These chemicals will clean different types of proteins out of your column. Isopropanol, ethanol, and non-ionic detergents are good at removing proteins that are hydrophobically bound to the column. If you need more information about non-ionic detergents that you can use for cleaning your column, this article is a great resource.

In contrast, sodium hydroxide (NaOH) will clean out precipitated or denatured proteins.

NaCl does a good job eliminating ionically-bound proteins from the beads.

By using a combination of these different types of cleaning solutions, you can make sure to thoroughly clean out all different types of proteins from your purification column. Typically, when I’m cleaning my resin, I will do 3 wash steps with 1.5 M NaCl, 1 M NaOH, and 70% ethanol. Of course, I rinse my column with dIH₂O in between these washes.

While these wash steps are pretty easy, one important point to keep in mind is that you will want to use a specific amount of contact time. Too fast, and your beads may not get thoroughly cleaned. And incubating too long with some of these cleaning solutions can damage your beads. See Table 1 for desired contact times for each of these cleaning solutions.

Table 1. Contact time for cleaning solutions

Cleaning Solution	Contact Time
1.5 M NaCl	15-20 minutes
1 M NaOH	1-2 hours (not overnight)
30% isopropanol	15-20 minutes
70% ethanol	15-20 minutes
0.5% non-ionic detergent in 0.1 M acetic acid	1-2 hours (not overnight)

Once you are done washing with these buffers, you will immediately rinse with a few column volumes of dIH₂O followed by a few column volumes with binding buffer, or some kind of buffer with a neutral pH around 7 to 8. This is because the pH of many of these stringent washes are quite extreme, so you want to reestablish a more neutral pH.

Nickel recharging buffer

We are now ready to add nickel ions back onto our cleaned agarose beads. To do this, we simply add 1.5 CVs of a 0.1 M nickel solution such as nickel chloride. Then rinse the excess metal out with several volumes of dIH₂O.

If you want to add a different metal such as cobalt, you can do that during this step. Cobalt has a different specificity and yield profile compared to nickel. So, if you wanted to switch out the type of metal on your agarose beads, using this protocol is a great way to do that.

That’s it, our column is now cleaned and recharged. If you had reused your column several times and its potency was tapering off, its binding capacity should now be restored as good as new.

The last step is that if you’re not going to use these beads for a while, go ahead and add a few column volumes of 20% ethanol for storage, and keep your beads in the fridge until you’re ready to use them. The cool temperature and 20% ethanol will prevent unwanted microorganisms from growing in your beads and contaminating your future protein purifications. Then, when you’re ready to start your next purification, just rinse the ethanol out with a few CVs of dIH₂O before getting started.

Can I regenerate my nickel beads forever?

You cannot regenerate your nickel beads forever. As a best-practice, you should be able to get roughly three to five purifications out of your nickel beads with proper cleaning and usage.

Your purification buffers and cleaning buffers will, over time, start to break down the agarose beads. If you notice a loss in binding capacity even after cleaning and recharging your beads, go ahead and discard that batch of beads and use new beads instead.

Should you even regenerate your nickel beads in the first place? It’s really a trade-off of money versus time.

The above protocol will take roughly half of a working day. It is not the most hands-on intensive protocol, so usually you can read a paper or design a new plasmid as you clean your beads in the background. But still, the time that you are regenerating your beads is time that you could be doing something else.

If you rarely use nickel beads, or use small volumes at a single time, it may not be worth the time investment to reuse them.

However, if you frequently used of nickel agarose beads, you may want to combine all of your used beads in a single jar, and occasionally strip, clean, and recharge them as a large batch that you can process all at once.

So, that’s how you can strip, clean, and recharge your nickel agarose beads to get more bang for your research buck. Keep in mind, you can’t clean and recharge your beads forever, but even reusing your agarose beads a few times is a smart, cost-saving decision.

If you want to learn more about nickel affinity protein purifications, or you’re ready to start purifying his-tagged proteins, then check out the Related Products and Related Resources sections below and see how GoldBio can help support your research program.