Protein L binds to the kappa form of antibody light chains and is used to purify antibodies and support immunoprecipitation (IP) experiments. Protein L recognizes human, mouse, and rat IgA, IgD, IgG, and IgM antibodies.


Protein L binds to the kappa form of light chains of human, mouse, and rat IgA, IgD, IgG and IgM antibodies (Figure 1). Just like Proteins A and G, Protein L is also useful for purifying antibodies and serving as an antibody binding site in immunoprecipitation (IP) experiments (Derrick & Wigley, 1994).By the way, check out this article if you need a quick reminder of antibody types and their overall structure.

protein L binding to the light chains of an antibody

Figure 1. Protein L binds to the kappa light chain of antibodies.

In regard to antibody purification, antibodies are molecules that are purified out of a complex mixture such as animal serum or cell lysate. Native Protein L itself was traditionally purified from the bacterium Peptostreptococcus magnus (Akerström & Björck, 1989). However, today’s recombinant Protein L is frequently expressed in, and purified from, Escherichia coli (Nilson et al, 1993).



protein L bound to antibody light chains

Figure 2. Protein L (green) bound to human IgM Fab light chains (orange). Heavy chains are purple (PDB: 1HEZ).


Affinity Purification of Antibodies

To purify antibodies, native or recombinant Protein L is conjugated to agarose beads. Usually, researchers don’t perform this step themselves. Rather, they buy premade Protein L agarose beads, such as the magnetic Protein L agarose beads that GoldBio sells (see link at the bottom of this article). Antibody-containing solutions are then loaded onto a Protein L column. After washing, the antibodies are then eluted by adding buffer with acidic pH (pH<3) (Figure 3).


Figure 3. Antibody purification. Antibodies bind to agarose beads with protein L (middle column). After washing (middle column), antibodies are eluted with an acidic pH elution buffer that disrupts the interaction between the antibody and protein L (column 3).


Antibodies have poor long-term stability in acidic conditions, so the eluted antibodies are then pH adjusted back to neutral values (~ pH 7 to 8).

These antibodies can be used for a number of applications, including helping purify other proteins in immunoprecipitation (IP) experiments.



Immunoprecipitation

Immunoprecipitation (IP) experiments use an antibody to bind to a particular protein and interrogate, or investigate, that protein’s molecular interactions.

Immunoprecipitation experiments work very similar to affinity chromatography, where researchers purify a protein based on a known interaction between two molecules, one of which is anchored to an agarose bead and the other fused to the molecule of interest. In this case, an antibody is bound by Protein L which is conjugated to an agarose bead, therefore being immobilized. Because antibodies are specific for certain molecules and proteins, it’s then used to capture a protein of interest within a mix.

Protein L beads are useful for IP experiments because they anchor the antibody. First, the antibody is loaded onto Protein L beads. Then, cell extract, or another complex biochemical mixture is loaded onto the antibody-loaded Protein L beads. That specific protein, and any of its interacting molecules, will stick to the antibody on the beads and can be eluted later. In contrast, proteins that don’t interact with the antibody-bound protein will flow through the beads before the elution (Figure 4).

overview of immunoprecipitation and protein L

Figure 4. Immunoprecipitation. Protein L agarose beads anchor 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 flow through the column without binding to the antibody or protein of interest (pink circles in final frame within the column).


This type of experiment is commonly used to discover and verify molecular interactions. For more information about IP experiments and how Protein L agarose supports these foundational experiments – check out this article!

That’s a quick overview of Protein L and how it is used in antibody purification and immunoprecipitation experiments. See the links below for related material about Protein L, and for more information about GoldBio’s Protein L agarose products.