“You young people view everything as single-use and disposable.” My Ph.D. advisor had just found out that I had not been cleaning and regenerating my affinity columns for protein purification.

In my defense, I was busy. Like, really, really busy. Supervising multiple undergrads on their research projects, teaching classes, writing my dissertation and articles for publication, purifying multiple proteins a week trying to keep my own research going. But, as usual, she was right. It was wasteful to throw perfectly good columns away and buy new ones when all I needed to do was clean and regenerate them and they would be as good as new.

Cleaning and regenerating agarose beads for protein affinity purification is a great way to keep your reagents in tip-top shape and stretch your research dollars. In this article, we’ll discuss how to clean and regenerate glutathione resin so you can successfully purify GST-fusion proteins over and over again.

Glutathione agarose beads are cleaned and regenerated by alternating several column volumes of: 1) 0.1M Tris-HCl pH 8.5, 0.5M NaCl, and 2) 0.1M sodium acetate pH 4.5, 0.5M NaCl. Additional, more stringent wash buffers are used to remove hydrophobically bound and precipitated proteins.


Table of Contents:

When to regenerate glutathione agarose resin?

What is glutathione resin regeneration?

Which buffers are used for glutathione agarose regeneration?

 

When to regenerate glutathione agarose resin?

Glutathione agarose beads are used to purify GST and GST-fusion proteins. Like many affinity purification steps, there are load, wash and elution steps to purify your protein (Figure 1).

Illustration of Glutathione Affinity Chromatography Steps

Figure 1. GST tagged fusion proteins bind to glutathione agarose (column 2) and after washing, are eluted with elution buffer with excess glutathione (column 3).


But there is actually an optional fourth step that you can do after you’ve collected your protein off of the column: washing and regenerating the beads so that you can use them again.

If you think you will be purifying the same exact protein again in the future, then you should 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.

If you’re going to use the beads again, but not for the same exact protein, then you will want to clean and regenerate your beads to make sure that you clean out any residual proteins from your previous purification(s).

Even if you’re purifying the same protein over and over, it is probably still a good idea to clean and regenerate your column about every five purifications, or earlier if you notice that the binding capacity of your beads is getting worse.

When to regenerate glutathione beads decision tree

Figure 2. Decision tree to show when to wash versus regenerate glutathione agarose beads.


What is glutathione resin regeneration?

Let’s dive into a quick nomenclature issue about regenerating agarose beads.

In the context of glutathione agarose beads, we can think of regeneration as a more stringent cleaning. For some other types of affinity purifications, like Nickel Beads and His-tags, regeneration means that we actually strip the nickel ion off of the beads and regenerate them by adding new nickel onto the beads.

For glutathione agarose, however, we don’t strip the glutathione and add it back on, so regeneration in this context is more like a really good cleaning that restores the original binding capacity to the glutathione beads.


Which buffers are used for glutathione agarose regeneration?

So, if you’ve determined that you should regenerate your glutathione agarose beads before your next purification, what buffers should you use for this job?

The most common buffers used to regenerate glutathione beads are alternating 10 column volume washes of:

  1. 0.1M Tris-HCl pH 8.5, 0.5M NaCl
  2. 0.1M sodium acetate pH 4.5, 0.5M NaCl

Repeat for a total of three washes with each buffer.


Often, the above wash combination will be all that you need. But sometimes, you might need a little bit more to really get all of the old protein out of there. In that case, pick a buffer or two from the following list, and wash with each of them:

  1. 1M sodium acetate, pH 4.0
  2. 0.1M sodium hydroxide
  3. 0.1M hydrochloric acid
  4. 70% ethanol
  5. 1% SDS
  6. 1% Triton X-100

Do not combine any of the chemicals from this list of more stringent cleaning agents together.

If you’re using more than one of these to wash your column, make sure you use them individually with a dIH2O wash in between so that the different components don’t come in contact with one another. Some of them are incompatible with each other and will precipitate which will clog your column.

These chemicals will get different types of proteins out of your column. Ethanol and detergents like SDS and Triton X-100 are good at removing proteins that are hydrophobically bound to the column.

In contrast, strong acids and bases like hydrochloric acid and sodium hydroxide, respectively, will clean out precipitated or denatured proteins.

The regeneration buffers will do a good job eliminating ionically-bound proteins from the beads. By using a combination of these different types of buffers, you can thoroughly clean out all different types of proteins from your purification column.


Table 1. Additional buffers for glutathione agarose resin regeneration.

Table of additional buffers for glutathione agarose resin regeneration.

NOTE: Rinse thoroughly with dIH2O after using any of these buffers.


While these wash steps are pretty easy, there’s a couple of important points to keep in mind.

The first point is to limit contact time. Many of the buffers in the additional buffer list like 0.1M sodium hydroxide, are rather harsh. You typically will want to be washing your beads with them for 1 hour, or less, of total contact time.

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 before long term storage of the beads.

Lastly, when you’re all done cleaning your beads, wash them with a few column volumes of dIH2O followed by a few column volumes of 20% ethanol, then store your beads in 20% ethanol in the fridge. The low temperature and 20% ethanol will prevent unwanted microorganisms from growing in your beads and contaminating your protein purification.

So, that’s how you can clean and regenerate your glutathione agarose beads to get more bang for your research buck. Keep in mind, these procedures won’t last forever, but even reusing your agarose beads a few times is a great move.

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