Have you heard of Protein A, Protein G, and Protein L? Maybe you know that you need one of them to purify antibodies or for your immunoprecipitation experiment, but you’re not sure exactly which one to use? If so, this is the right article for you.

The key difference between Proteins A, G, and L is that they bind to different types of antibodies. All bind to human and mouse IgG antibodies, Protein A and G also bind to IgG antibodies from different species, and Protein L also binds to IgA, IgD, and IgM antibody subtypes.

In this article we will cover 3 key similarities and differences between Protein A, Protein G, and Protein L. Hopefully learning more about these 3 important proteins will help you decide which one is best for your experiments.

Article Table of Contents

Key Similarities for Antibody Binding

Proteins A, G, and L bind to antibodies

They support immunoprecipitation experiments

Proteins A, G, and L are bacterial proteins

Key Differences Between Protein A, Protein G and Protein L

Proteins A, G and L interact with different antibody subtypes

Protein A and G bind to heavy chains, Protein L binds to light chains

They come from different species

Choosing Between Proteins A, G, and L

Related Products

References

Key Similarities for Antibody Binding

To understand why Protein A, G, and L bind to different types of antibodies, let’s go over key similarities between these Proteins that impact antibody binding:

  • Proteins A, G, and L bind to antibodies
  • They support immunoprecipitation experiments
  • Proteins A, G, and L are bacterial proteins


Proteins A, G, and L bind to antibodies

Proteins A, G, and L all bind to antibodies (Figure 1). This feature is leveraged to affinity purify antibodies and for use in immunoprecipitation experiments. Often, researchers use agarose beads that have Protein A, Protein G, or Protein L conjugated onto the surface for these purposes.



They support immunoprecipitation experiments

Since Protein A, Protein G, and Protein L bind to antibodies, this makes them a useful reagent for immunoprecipitation experiments. Immunoprecipitation is when an antibody to a particular protein is used to grab that protein out of a complex biochemical mixture, like a cell lysate, for example.

Protein A binding to the heavy chain of an antibody

Figure 1. Left, cartoon model of Protein A interacting with an antibody. On the right is the molecular structure of Protein A (green) interacting with human IgM antigen binding fragment (Fab) via the heavy chains (purple). Light chains are colored orange (PDB: 1DEE).


Immunoprecipitation may be used to quantify that protein in different conditions, such as different cell types. Or it may be used to investigate what other kinds of molecules interact with the protein that the antibody binds to. (Figure 2). Protein A, G, and L agarose beads are extremely useful for this later use.

Conceptual illustration of immunoprecipitation

Figure 2. 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 (blue diamond). If the protein of interest interacts with another protein (green oval), the nature of that interaction will capture those interacting proteins as well. Contaminating proteins (pink circles) 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 similarly to Protein A or L instead.



Proteins A, G, and L are bacterial proteins

Proteins A, G, and L are all bacterial proteins. While researchers use them to purify antibodies and support immunoprecipitation experiments, in their natural environment these proteins bind to antibodies as a defense mechanism.

By binding to antibodies, these proteins fight against being phagocytosed, or eaten, by the host’s B cells (Kastern et al, 1990; Kobayashi & DeLeo, 2013). Avoiding being destroyed contributes to these bacteria’s survival, usually to the detriment of the host.

Just because Proteins A, G, and L all bind to antibodies, support immunoprecipitation experiments, and are bacterial proteins doesn’t mean that they are exactly the same though. Next, let’s dig into some of the key differences between these proteins.


Key Differences Between Protein A, Protein G and Protein L

So far, we’ve covered some key similarities among Proteins A, G, and L – but there are important differences too, and these differences can help you determine the most appropriate choice for your experimental needs.

Let’s dive into some of the ways that Protein A, Protein G, and Protein L are different from one another:

  • They interact with different antibody subtypes
  • Protein A and G bind to heavy chains, Protein L binds to light chains
  • They come from different species

Proteins A, G and L interact with different antibody subtypes

There are five classes or isotypes of antibodies in humans and related mammals: IgA, IgD, IgE, IgG, and IgM. You noticed that each type starts with Ig, and that’s short for immunoglobulin – antibodies are a common form of immunoglobulin.

The different antibody classes are found in different locations in our bodies. They bind to distinct antigens and perform different biological functions. See Table 1 for more information on the locations, antigens, and biological functions of different antibody classes.


Table 1. Antibody Classes

Class

Heavy Chain

Unique Features

IgA

a

Prevents pathogen colonization in saliva, tears, breast milk, and on mucosal membranes.




IgD

d

Found on naïve B cells that have not yet been exposed to antigens.




IgE

e

Protect against parasitic worms, also involved in allergies and asthma.




IgG

g

Detect pathogens in blood and extracellular fluid.




IgM

m

Initiates inflammatory reactions to neutralize and clear pathogens.


Protein A, G, and L have distinct binding specificities for different antibody classes from different species. For example, Protein A and Protein G are both good for binding to IgG antibodies, but they have different preferences for species: Protein A binds to pig, dog, cat, and guinea pig; Protein G binds to goat sheep, donkey, cow and horse; and both Protein A and Protein G bind to human, mouse, and rabbit.

Protein L also binds to human and mouse IgG as well as human, mouse and rat IgA, IgD, and IgM antibodies. That’s a lot to remember – check out Figure 3 and Table 2 (toward the end of this article) for quick references on all of that!

diagram of the similarities and differences of protein A, protein G, and Protein L


Protein A and G bind to heavy chains, Protein L binds to light chains

Proteins A and G bind to the heavy chains of antibodies, whereas Protein L binds to light chains. Specifically, Protein L binds to the kappa form of light chains, which is part of the reason it binds to different subtypes of antibodies relative to Proteins A and G. For example, Protein L is better at binding to IgA, IgD, and IgM antibodies through kappa light chains (Figure 3 and Table 2).

Earlier in Figure 1, you saw the structure of Protein A binding to an antibody. Look back at Figure 1 and notice how it’s the heavy chain, colored in purple that makes up all of the interaction surface with Protein A.

Now let’s consider Protein L. Notice in Figure 4 how it’s the light chains, colored in orange, that bind to Protein L.

Protein L molecular image

Figure 4. Protein L (green) bound to human IgM Fab light chains (orange). Heavy chains are purple (PDB: 1HEZ).


They come from different species

Remember how we mentioned that Proteins A, G, and L all come from bacteria? While that’s true, they each come from different bacterial species. Protein A is from Staphylococus aureus, Protein G is from type C and G Streptococcal bacteria, and Protein L is found in Peptostreptococcus magnus.

To generate Protein A-, G-, and L-conjugated agarose beads, each of these proteins was originally purified from their native bacterial species, though there are now recombinant forms of these proteins that are expressed in less virulent bacteria strains to increase lab safety (Björck et al, 1987; Derrick & Wigley, 1992; Graille et al, 2001; Hjelm et al, 1972; Kittler et al, 2022).



Choosing Between Proteins A, G, and L

Ok, now that we’ve discussed key differences between Proteins A, G, and L, let’s discuss how to choose between these proteins. The key point here is that choosing between Proteins A, G, and L will depend on which type of antibody that you’re working with.

For example, if you’re using a human or mouse IgG antibody, Protein A, G, or L may work well, and you could try all of these if you want to see which one works the best. But here is a guide to narrow down your choice when using certain types of antibodies:

  • For human, mouse, and rat IgA, IgD, and IgM antibodies, Protein L is likely the best choice.
  • For rabbit IgG antibodies either Protein A or Protein G would probably work well, and you could compare them head-to-head to see which one works best.
  • Protein A is probably your best choice for pig, dog, cat, and guinea pig IgG antibodies.
  • Protein G will likely work best for goat, sheep, donkey, cow, and horse IgGs.

That’s a lot of information to keep track of! Again, see Figure 3 and Table 2 for convenient summaries of this information.

The previous paragraph is full of qualifiers like, “probably” and “likely.” That is because these are not hard rules that will be absolutely true for every antibody. Rather, you should view these suggestions as good starting points for you to figure out which protein binds the best to your unique antibody.

Table 2. Antibodies recognized by Proteins A, G, and L

Species

Subtypes

Protein A / G / L

Human & Mouse

IgG

A, G, & L

Human, Mouse, & Rat

IgA, IgD, & IgM

L

Rabbit

IgG

A & G

Pig, Dog, Cat, & Guinea Pig

IgG

A

Goat, Sheep, Donkey, Cow, & Horse

IgG

G



Now you know 3 key similarities and differences between Proteins A, G, and L. If you’re looking for Protein A, Protein G, or Protein L agarose beads to purify antibodies or conduct immunoprecipitation experiments see below for links to great GoldBio products. Also, if you want more information about Protein A, G, and L, check out the links throughout this article to many of our other articles in this general research area.