Have you ever seen a cartoon where a huge magnet is used to pull objects into the sky? Usually, the magnet is intended to secretly steal some treasure or incapacitate a foe. Invariably it sucks up everything in sight, bringing the protagonist far more than they bargained for.

The mental image of things flying through the sky is exactly what I want you to have in mind as we talk about protein purification using magnetic agarose beads. These beads have ligands that bind to tagged proteins or antibodies. Also, the beads are magnetic, meaning when a magnetic force is applied, they fly through the solution, thereby purifying the attached protein from the rest of the molecular milieu.

Magnetic agarose beads are used to purify affinity-tagged proteins and antibodies. The magnetic feature rapidly separates the beads from the rest of the solution when a magnetic force is applied.

In this article we’ll discuss magnetic beads, the different ligands available for protein purification, and how these captivating beads are used for protein purification.

Article Table of Contents:

Magnetic Agarose Beads

Affinity Ligands for Magnetic Agarose Beads

Protein Purification with Magnetic Agarose Beads

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References


Magnetic Agarose Beads

Magnetic agarose beads are essentially the same as regular agarose beads except that they’re also magnetic. Nanoscopic magnetic particles are included during agarose bead formation, and becomes embedded in the beads. These particles are usually made out of iron and give the beads their magnetic property (Figure 1)(Wang et al, 2020).

Diagram of magenetic bead with ligands and particles labeled

Figure 1. Magnetic agarose beads have magnetic particles (orange) and ligands (blue) attached for protein purification.


The other feature of GoldBio’s magnetic agarose beads are ligands that bind to tagged proteins and antibodies (Figure 1). Next, let’s talk a little bit more about what exactly those ligands are and what they do.



Affinity Ligands for Magnetic Agarose Beads

Before we discuss more about how magnetic beads are used, let’s dive into the different types of magnetic agarose beads that you can get from GoldBio. These beads are used for the affinity purification of tagged proteins and antibodies (Table 1).

Table 1. Gold Bio Magnetic Agarose Beads

Magnetic Beads

Tag / Feature

Nickel NTA

His-tag

Protein A

Antibodies – Heavy Chain

Protein G

Antibodies – Heavy Chain

Protein L

Antibodies – Light Chain

Nickel NTA beads bind to his-tagged proteins. His-tags are repeats of the amino acid Histidine, usually at least 6 Histidine residues long. By the way, the three-letter abbreviated form of Histidine is “His” which is why these are referred to as “His-tags.”

The His-tag is added onto a protein of interest to help purify that protein. The Histidine side chains coordinate Ni, or other similar transition metals (Figure 2). Imidazole, which you can see is the side chain of Histidine, is added to elute His-tagged proteins from the column. See this article if you want to learn more about His-tags and how they’re used to purify proteins.

The imidazole side chains of histidine residues in the His-tag (purple) bind to Ni2+-conjugated agarose beads

Figure 2. The imidazole side chains of histidine residues in the His-tag (purple) bind to Ni2+-conjugated agarose beads. Free imidazole (blue) is added in excess to elute His-tagged proteins from the Ni2+ beads.


Protein A, Protein G, and Protein L are three bacterial proteins that bind to antibodies. Proteins A and G bind to the heavy chains of antibodies, whereas Proteins L bind to light chains. For more similarities and differences about Proteins A, G, and L, see this article.

A buffer with acidic pH is then used to elute the antibody by disrupting the electrostatic interactions between Protein A, G, or L and the antibody (Figure 3).

Antibodies (purple and orange) bind to Protein A, G, or L (green) in part through electrostatic interactions (left).

Figure 3. Antibodies (purple and orange) bind to Protein A, G, or L (green) in part through electrostatic interactions (left). An acidic elution buffer disrupts this interaction by changing protein charge (right).


Protein Purification with Magnetic Agarose Beads

Ok, now that we’ve discussed the different types of magnetic agarose beads, let’s get to how magnetic beads are used to purify proteins. The magnetic bead that will purify your protein of interest (Ni NTA for His-tagged proteins or Protein A, G, or L for antibodies) is added into a biochemical mixture that includes your protein of interest. Usually that is just the cell lysate or secretion from whichever type of cell you used to express your protein of interest.

After incubating the beads with this mixture, which allows the interaction to form between the ligand on the agarose bead and your protein of interest, you’ll expose your sample to a magnet. The magnetic force will draw the agarose beads towards the magnet, whereas the unbound proteins will remain in solution (Figure 4).

magnetic beads in tube on a magnetic tube holder

Figure 4. The magnetic beads with target protein (green rectangle) bound are drawn toward the magnet whereas the other proteins (orange and pink) remain in solution.

While the magnetic beads and bound molecules are drawn up against the side of your tube, you will pipette out the unbound solution. In order to wash the beads, new buffer is added to the tube. The tube is moved away from the magnetic force and the new buffer resuspends the beads and attached proteins. Then, the magnetic force is reapplied and the wash buffer is pipetted out of the tube. This type of wash step is usually repeated once or twice to thoroughly wash away any residual unbound molecules (Figure 5).

Pipetting out contaminating proteins

Figure 5. Pipetting out contaminating proteins (orange and pink).


The elution buffer is then added to the tube, the magnetic force is applied again, and this time you’ll pipette out your protein of interest (Figure 6).

eluting the protein of interest from magnetic beads


Figure 6. Eluting the protein of interest (green). A solution is added to disrupt the interaction between the ligand and the protein of interest (left). Then the protein of interest is pipetted out of the tube (right).


Since the cartoon may not fully reveal what this process looks like in real life, check out Figure 7 to see what the magnetic beads actually look like.


Figure 7. Real-life images of protein purification with magnetic agarose beads.



So that’s how magnetic agarose beads are used to purify proteins. People often find that the magnetic versions are more convenient for doing small-scale screening of several versions of a protein in parallel, as compared to regular agarose beads.



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