How to Prepare Glutathione Elution Buffer

If you’re purifying GST or a GST-fusion protein with glutathione agarose beads, then a key step in this process is making and using glutathione elution buffer. Making glutathione elution buffer isn’t hard, but there are a few additional things to consider relative to elution buffers for other types of affinity purifications:

To make glutathione elution buffer, add reduced glutathione powder to your buffer at a final concentration of 10 to 20 millimolar (mM) within an hour or two of when you will use the buffer. Glutathione is an acid so you will likely need to adjust the pH of your buffer before using it.

In this article, we’ll discuss how to make glutathione buffer and some important considerations to keep in mind as you’re making it such as adjusting the pH, reduced versus oxidized glutathione and more.

Table of Contents:

Making glutathione elution buffer

How to adjust the elution buffer’s pH

Reduced vs oxidized glutathione

Compatibility with other buffer components

Making glutathione elution buffer

Glutathione affinity chromatography is a type of affinity purification used to purify GST and GST-fusion proteins (Figure 1).

Figure 1. Glutathione affinity chromatography for the purification of GST-fusion proteins.

Let’s get into the meat of this article: making glutathione elution buffer. It’s really simple to make glutathione elution buffer. It just takes a few steps:

• Add reduced glutathione to a final concentration of 10-20mM to your buffer of interest.
• Gently invert or add a stir bar to solubilize the glutathione.
• Adjust the pH back to the intended value using a concentrated base, if necessary.

I’ve already mentioned “buffer” or “buffer of interest” a couple of times without specifying which buffer you should use. What makes a good elution buffer? One of the great things about glutathione agarose beads and glutathione is that they work well with many different buffer components, as I will discuss in the last section of this article.

When I’m purifying GST-fusion proteins, I simply add the glutathione to whatever my binding buffer is to make buffer preparation easy. In our GST purification protocol, we suggest using 1x PBS, pH 7.4 as the binding buffer. So, if that is the binding buffer that you’re using, just add the glutathione to that and it will work great as an elution buffer.

The other, and probably more important consideration for your elution buffer is: what are you going to do with your protein next?

For example, if you’re going to additionally purify it by ion-exchange chromatography, then you might want to elute in a buffer with low salt. If you’re going to use the protein in a particular assay, then elute it in a buffer that is compatible with that assay.

Again, one of the nice features of glutathione agarose beads is their flexibility with a wide range of buffer components.

Learn More About:

What glutathione agarose is

Solubility tags

How to adjust the elution buffer’s pH

Glutathione is an acid. Glutathione is the tripeptide glutamic acid-cysteine-glycine (Figure 2). As you can probably tell from the name, glutamic acid makes glutathione an acid, and there is not a corresponding basic amino acid (lysine or arginine) in glutathione to neutralize it.

If you want to learn more about the acid and base chemistry of amino acids, check out this article where we go into great detail on the subject.

Figure 2. The glutamic acid (pink) in glutathione makes it acidic.

Since glutathione is an acid, you’ll want to check the pH of your buffer after you add it.

If you’ve used a high concentration of pH buffer, such as 50mM Tris pH 8, then the addition of 10-20mM glutathione may not significantly change the pH of your buffer. It is always better to check to make sure though.

If you’re using a more normal concentration of pH buffer, say 20mM Tris pH 8, then adding 10-20mM glutathione will make your elution buffer more acidic. Most proteins are not stable in acidic pH, so you will want to adjust your pH back to the desired value using a concentrated base such as sodium hydroxide before using your elution buffer.

The first thing you would do after solubilizing glutathione in your elution buffer is measure the pH. If it is more than 0.1 or 0.2 pH units away from your desired pH, then you’ll want to use concentrated NaOH to adjust the pH of your elution buffer.

If you have a pH meter, leave the probe in your elution buffer with a stir bar rotating, and add one or two drops of concentrated sodium hydroxide at a time, then give the solution a few seconds for its pH to reach an equilibrium. Repeat this process until your buffer is the desired pH.

Alternatively, if you’re using pH strips, remeasure your pH after every one or two drops of NaOH is added.

Reduced vs oxidized glutathione

Glutathione comes in reduced and oxidized forms. The reduced form binds more tightly to GST, so this is the form you want to include in your elution buffer.

Over time, oxygen in the air will convert reduced glutathione into the oxidized form (Figure 3).

Figure 3. Atmospheric oxygen converts reduced glutathione (left) to oxidized glutathione (right). The oxidized bond between glutathione molecules is colored light blue.

This is why you want to add the glutathione to your elution buffer right before you use it. You’ll still have plenty of the reduced form of glutathione in your buffer within a few hours of making it. If it’s the next day, however, you’re better off making a new buffer with fresh reduced glutathione instead of using your old one.

See this article if you want to learn more about the differences between these different forms.

Compatibility with other buffer components

Glutathione, the key component in GST elution buffers, and glutathione agarose beads are compatible with a wide variety of commonly used buffer components including reducing agents, detergents, buffers, and denaturing agents (Table 1).

Table 1. Chemical compatibility with glutathione and glutathione agarose beads.

Keep in mind that while glutathione and glutathione agarose beads are compatible with all of these components, you need to ensure that your GST tag is folded and capable of binding to glutathione.

So, for example, 8M urea or 6M guanidine HCl would likely denature GST, preventing the GST-glutathione interaction that serves as the foundation for this purification. So if either of these denaturing agents are required for your purification, you’ll want to carefully optimize the concentration that you need, or consider using a different type of affinity tag.

That’s how to prepare glutathione elution buffer for the purification of GST and GST-fusion proteins. Below we have links to reliable GoldBio products and to related articles, purification protocols, and troubleshooting guides. If you’re ready to start purifying GST-fusion proteins, or need more information about this powerful experimental approach, check those links out!