How to Perform Immunoprecipitation with Protein A, G, or L Agarose Beads (2025)

Immunoprecipitation (IP) is a powerful and frequently used technique that utilizes antibodies to isolate a particular protein of interest out of a complex biochemical mixture such as lysed cells. “Immuno” refers to the antibodies being used, which are part of the immune system. “Precipitation” indicates how this approach “pulls” a protein of interest out of solution and onto a solid stationary support.

Performing immunoprecipitation with Protein A, G and L involves Protein A, G or L agarose beads binding to antibodies and immunoprecipitating the protein that antibody recognizes. Immunoprecipitation is used to evaluate a protein or to identify other molecules that interact with that protein.

Protein A, G, and L agarose beads are commonly used reagents for IP experiments, and they serve as the solid stationary support.

In this article, we’ll discuss how these agarose beads support IP experiments and cover a few common uses for this technique.

Article Table of Contents

Immunoprecipitation

Using Protein A, G, or L?

Loading the Agarose Beads

Eluting the Agarose Beads

Analyzing IP Experiments

Related Products

References

Immunoprecipitation

To provide an overview of immunoprecipitation, let’s first discuss Protein G as an example. In the next section we will discuss some relevant differences between Proteins A, G, and L, but in terms of setting up an IP experiment they are conceptually the same.

To start with, we’ll have agarose beads with Protein G conjugated to them (Figure 1). If you want more information about how agarose beads are made, or how ligands like Protein G are conjugated to them, check out those articles.

Figure 1. Protein G (green) conjugated to an agarose bead (blue). Once in a column, this works as a support for immunoprecipitation.

Then you’ll add the antibody you are using for the IP experiment. This will bind to the Protein G-conjugated agarose bead (Figure 2). Note that you’ll want to use Protein A, Protein G, or Protein L, depending on which type of antibody you’re using, which we’ll cover in the next section. But for now, let’s continue with Protein G for illustrative purposes.

Figure 2. Protein G is bound to agarose beads in solution (frame 1). Antibodies (pink) are added in order to bind with Protein G (frame 2). Protein G binds to the heavy chains of this antibody (frame 3 and callout bubble).

Figure 3. Immunoprecipitation. Protein G agarose beads anchor IgG antibodies (first 2 frames). The antibodies are specific for a certain antigen and will bind to them, thereby capturing the protein of interest (orange). If the protein of interest interacts with another protein (purple), the nature of that interaction will capture those interacting proteins as well. Contaminating proteins (Lime) flow through the column without binding to the antibody or protein of interest in the final frame within the column. While this figure is shown with Protein G, it would work the same way for Protein A or L instead.

Next, you’ll introduce the protein of interest that your antibody binds to. This will likely be in the context of a complex biochemical mixture, such as cell or tissue extract.

What you do next will depend on what kind of information you want out of the experiment, but likely you’ll be eluting the antibody, your protein of interest, and anything else that is bound to it, from the Protein G column.

In Figure 3 above, you see that the protein of interest is orange. However, the purple protein interacts with the protein of interest. This means that when you elute your antibody, it’s not just your protein of interest that will come along with it, but also the interacting protein (purple).

Using Protein A, G, or L?

Proteins A, G, and L all bind to antibodies. This feature is used to affinity purify antibodies and for immunoprecipitation experiments. But Proteins A, G, and L have different specificities, or preferences, for the types of antibodies that they bind to (Table 1).

Table 1. Protein A, G, L Specificity

One important difference is antibody subtype. Different subtypes localize to different locations in our bodies and perform distinct functions (Table 2). Protein A and G tend to be better at binding to IgG antibodies, whereas Protein L is better at interacting with IgA, IgD, and IgM antibodies.

Another relevant difference is what species the antibody comes from. While both Protein A and G bind well to IgG antibodies, Protein A binds better to IgGs from pig whereas Protein G is better for goat IgGs.

Consider these specificities when deciding between Protein A, G, and L for your IP experiment.

Table 2 Antibody Classes

Loading the Agarose Beads

We discussed loading antibodies and your protein of interest onto the agarose beads. One important consideration here is that you want to maintain buffer conditions that promote the interaction between Protein A, G, or L (whichever one you’re using) and the antibody, otherwise the antibody and your protein of interest will slip right off the column.

To understand more about buffer conditions, we have a really detailed resource here that may really help. But the take-home point is that you’ll want to maintain neutral pH and normal to low salt concentrations during the loading step. Something like PBS, pH 7.4 works well for these purposes.

Eluting the Agarose Beads

To analyze your IP experiment, you’ll usually want to elute the antibody. The most common method for eluting antibodies from Protein A, G, or L is to use an acidic elution buffer which disrupts the electrostatic interactions holding that interaction together (Figure 4B). Other, less common elution methods include a basic elution buffer or high salt which, in the end, disrupts the interaction in the same kind of way (Figure 4C-D). For more information about eluting antibodies from Protein A, G, and L agarose beads, see this article.

Figure 4 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 (D) the salt “screens” the interaction between the two proteins.

Analyzing IP Experiments

A couple of common uses for IP experiments is to determine the relative expression of a protein of interest in different cell types, or to search for other molecules that interact with your protein of interest.

First, let’s consider analyzing the expression of your protein of interest in different cell types. Perhaps you’re working with a novel protein, and in order to help determine its function you want to know what kind of tissues it is expressed in. You could perform several IP experiments, in parallel, using cell extract from different cell types for each IP (Figure 5).

Figure 5. IP to determine the relative amount of your protein of interest in different tissues. Top purple band is the antibody and the lower blue band is your protein of interest. In this hypothetical example the protein of interest is most highly expressed in the brain.

Additionally, if you want to know what other molecules interact with your protein of interest, you can use IPs to help figure that out. Figure 6 shows a hypothetical SDS-PAGE gel representing such an experiment. In this type of experiment, after loading the antibody and protein of interest onto the Protein A/G/L column, you would then flow over cell lysate to see which other proteins interact with your protein of interest.

By knowing the size of your antibody and your protein of interest, you can deduce which other proteins are interacting with your protein of interest. Using a technique such as mass spectrometry would help you identify those interacting proteins (Greenblatt et al, 2024). It’s a good idea to have an antibody-only control (left lane in Figure 6), so you can decipher whether these new proteins interact with the antibody itself, or your protein of interest.

Figure 6. IP looking for interacting proteins. Antibody and protein of interest are the same as in Figure 5. And now there are three interacting proteins (different shades of green bands) that interact with the protein of interest (right lane), but not the antibody alone (left lane).

The above example is looking at protein-protein interactions, but IPs are also used to identify other types of interactions like proteins binding to DNA. A specific type of IP, called ChIP (Chromatin IP), is used to see where in the genome DNA-binding proteins interact, which is an important step in gene regulation (Farnham, 2009). As you can tell, IPs are a really versatile technique that help uncover loads of novel biology!

So that’s how Protein A, G, and L agarose beads are used for immunoprecipitation experiments. If you want to know more about these versatile beads – check out some of our related articles linked throughout and the product websites below.