If you’re using Protein A, Protein G, or Protein L agarose beads to purify antibodies, then a key step in this process is eluting the antibodies from the beads. While there are several ways to disrupt antibodies’ interactions with Proteins A, G, or L during the elution step, the most common method is to use an acidic pH buffer.
Antibodies are eluted from Protein A, Protein G, and Protein L agarose beads using an acidic elution buffer such as tris-glycine or citric acid in the pH ~ 2.5 – 4.0 range. The pH is quickly neutralized following elution because antibodies are not stable long term in acidic conditions.
In this article we’ll discuss a variety of methods for eluting antibodies from Protein A, Protein G, and Protein L agarose beads, and why acidic pH buffer is usually used for this purpose.
Article Table of Contents
Additional References and Protocols
Antibodies are frequently purified using Protein A, Protein G, or Protein L agarose beads. For the purposes of this article, it doesn’t really matter if you’re using Protein A, G, or L agarose beads, as the elution step is basically the same for all of them. However, if you want to learn more about which type of agarose bead is the best for the antibody that you’re purifying, then this article and this article cover that choice in more depth.
A key step in the purification process is eluting the antibodies from the agarose beads after other contaminating proteins have already been washed away (Figure 1). Let’s dive into different elution buffers and discuss why an acidic buffer is a frequently used option.
Figure 1. Antibody
purification. Antibodies bind to agarose beads conjugated with interacting
partner molecules such as protein A, G, or L (column 2). After washing,
antibodies are eluted with an acidic pH elution buffer that weakens the
interaction between the antibody and protein A (column 3).
Elution Options
There are essentially four options for eluting antibodies from Protein A, G, and L agarose beads:
- acidic buffer
- basic buffer
- ionic strength
- denaturing conditions
These four options all disrupt electrostatic interactions between Protein A, G, L and antibodies (Figure 2A), though they each use different mechanisms to do so.
Acidic Buffer
Acidic buffers, such as tris-glycine or citric acid in the pH ~ 2.5 – 4 range, are very commonly used to elute antibodies from Protein A, G, and L agarose beads. The acidic buffer changes the charges on the antibodies and on Protein A, G, or L, thereby weakening the interaction between them (Figure 2B).
By the way, for more information about pH and how protein charge is dependent on pH – see this article and this article.
Basic Buffer
Similar to acidic buffers, basic buffers weaken the interaction between antibodies and Protein A, G, and L by changing the protein charges (Figure 2D). Common basic buffers include triethylamine and ammonium hydroxide in the pH 10 – 11.5 range.
Figure 2. Antibodies (purple and orange) bind to Protein A, G, or L (green) in part through electrostatic interactions (A). Acidic (B) and basic (C) elution buffers disrupt this interaction by changing protein charge, whereas in high ionic strength buffers (C) the salt “screens” the interaction between the two proteins.
Ionic Strength
Instead of changing the charge on Protein A, G, or L and the antibodies, another way to disrupt the electrostatic interactions between the interacting proteins is by adding a high salt buffer (Figure 2C). Examples of high salt buffers include 2 to 5 molar lithium chloride, potassium chloride, or sodium thiocyanate.
Denaturing Conditions
Denaturing buffers use harsh reagents such as urea, guanidine HCl, and SDS to denature the structure of Protein A, G, or L and the antibodies to disrupt the interactions.
While all of these elution methods are relatively harsh, this is the most extreme and destroys any functional antibodies, so this is not a good method when wanting to use antibodies for a downstream purpose.
Since most of the time you’re purifying the antibody to use for another experiment, this harsh elution condition is rarely used. If your downstream purpose doesn’t require native, functional antibodies – for example, you were just quantifying antibody levels from different conditions – then denaturing elution would work just fine for that purpose.
Eluting with Acidic Buffer
As mentioned, all of these elution methods are relatively harsh. These buffers don’t just change the interaction between Protein A/G/L and the antibody, they also change interactions within each protein and destabilize their fold or structure. Native and functioning antibodies can be retrieved from acidic, basic, and ionic elutions with proper handling, however. In contrast, denaturing agents destroy the native fold of the antibodies and should not be employed anytime you are purifying the antibody to use for downstream applications.
Time is of the essence here, and that is a big part of why acidic or basic buffers are preferred over ionic buffers. The pH of the elution solution can be rapidly adjusted to a neutral pH that is more stable for the antibodies. In fact, you can pre add the neutralization buffer into the elution tubes so that the pH will be neutralized as soon as it elutes off the column. Meaning that the antibodies are only exposed to destabilizing extreme pHs for seconds to maybe a few minutes depending on the size of your column.
Conversely, for high ionic solutions one would either need to rapidly dilute the solution, which is fast but would require a time-intensive reconcentration step downstream or dialyze the solution which is slow and would prolong antibody exposure to the destabilizing high salt conditions. This means that you either add hours to days to your purification protocol or the antibodies are exposed to destabilizing high ionic strength solutions for many hours while the salt slowly dialyzes away.
Acidic pH elution is usually tried as a first choice over basic elution buffers because it works for a wider variety of antibodies and tends to give higher yields for more antibodies. A basic pH can work better for some antibodies and may be tried if the acidic pH elution doesn’t work for a particular antibody.
Interestingly, basic pH buffers are also used as a wash step for many antibodies (Imura et al, 2021). While this basic pH wash helps improve the stability of the antibodies later eluted with an acidic pH, its use as a wash step highlights the poor elution properties of a basic pH for many antibodies.
So those are the options for eluting antibodies from Protein A, G, and L agarose beads, and the reasons why people usually use an acidic pH for this purpose. See the additional protocols and references below for more information on antibody purification using Protein A, Protein G, and Protein L agarose beads.
Additional References and Protocols
Protein A and Protein G Troubleshooting Guide
Affinity Comparison of Protein A and Protein G for Various Antibody Classes
