How to Use Magnetic Agarose Beads to Purify Antibodies: A Step-by-Step Protocol

This article provides a step-by-step protocol for purifying antibodies using Protein A, Protein G, or Protein L magnetic agarose beads.

Protein A, G, and L magnetic agarose beads purify antibodies by drawing them towards a magnetic force. This magnetic process pulls antibodies to the side of a tube, separating them from the surrounding solution during the load, wash, and elution steps.

Regular and magnetic agarose beads are both used to purify proteins and antibodies. Previously we provided a protocol for purifying antibodies with regular agarose beads and compared regular and magnetic agarose beads – so check out those articles if you’re interested in learning more about these subjects.

Article Table of Contents:

Purification

Materials

Prepare the beads

Rinse Step

Load

Wash

Elute

Neutralization

Analyzing your Purification

Additional References and Protocols

References

Purification

This purification protocol describes how to purify antibodies using the magnetic versions of GoldBio’s Protein A-, Protein G-, or Protein L-conjugated agarose beads. First, we’ll prepare the beads, then we’ll load our antibody-containing solution, next we’ll wash the beads, and finally we’ll elute our antibodies off of the beads and pH neutralize the eluted antibody solution (Figure 1).

Figure 1. Purification of antibodies. Antibodies bind to agarose beads conjugated with interacting partner molecules such as protein A, G, or L (tube 1). Those magnetic beads are drawn to the side of the tube closest to the magnet (blue rectangle) on the tube holder. After washing away contaminating proteins (tube 2), antibodies are eluted with an acidic pH elution buffer that weakens the interaction between the antibody and protein A (tubes 3 and 4).

Materials

• Microcentrifuge tubes, or another tube of appropriate size
• Magnetic tube holder that fits your selected tubes
• Magnetic agarose beads such as Protein A, Protein G, or Protein L
• Tube rotator or agitator
• Loading buffer such as PBS
• Elution buffer such as Glycine
• Neutralization buffer such as Tris Base
• SDS-PAGE gel and running buffer

Prepare the beads

GoldBio’s (and most companies’) agarose beads are delivered as a slurry in 20% ethanol to keep the beads sterile. If you’re planning on reusing the beads for additional purifications, it is a good idea to leave the beads in 20% ethanol when you’re done with this purification. To start a purification, you’ll want to get rid of the ethanol before equilibrating the beads in loading buffer.

The magnetic beads are in a 50% slurry, meaning half of the volume is agarose beads and half is the 20% ethanol solution. So, if you want an agarose bead volume of 0.1 mL, for example, then you should load 0.2 mL of slurry into the tube. The binding capacity and expected amount of expressed antibody will help you determine the volume of agarose beads that you should use for your purification.

Rinse Step

1. Invert the bead slurry in its container to homogenize the bead mixture and pipet the desired volume into your tube.
2. Expose the tube to a magnet to draw the magnetic beads to the side of the tube.
3. Pipet out the 20% ethanol solution and discard, being careful not to disturb the beads on the side of the tube.
4. Remove the tube from the magnetic field, add deionized water, close the tube and gently invert it a few times to rinse the beads.
5. Expose the tube to the magnet and carefully pipet out the water.
6. Repeat the water rinse step 2-3 more times, then another 2-3 more times with your loading buffer.

You can discard all liquids collected during this step because you won’t need them anymore.

In the above steps we use water and then a loading buffer to rinse the beads. Table 1 has frequently-used buffers, including a loading buffer, that work well for most antibodies. Also, see this article if you want a deep dive regarding buffers for purifying antibodies with Protein A, G, and L agarose beads. There are many different kinds of buffers you would be using, just make sure whatever your antibody solution is in is a pretty good buffer match with the loading buffer that you’re using here.

The beads are now ready for loading your antibody solution!

Load

1. Set aside a very small volume (~15 mL) of the antibody solution for further analysis.
2. Add the rest of the antibody solution by gently pipetting into the tube.
3. Put your tubes on a rotator or agitator on a gentle setting for 30 minutes to an hour, enabling sufficient time for Protein A, G, or L to bind to the antibodies.
4. Place the tube in the magnetic field, thereby drawing the agarose beads to the side of the tube.
5. Gently pipet out the remaining liquid, being careful not to disturb the beads on the side of the tube. Save this “flow-through” sample for further analysis.

An aside on nomenclature: the ‘flow-through” terminology is in reference to the more classical gravitational columns in which contaminating proteins flow through the column while the protein of interest binds . A more technically correct term for magnetic bead purifications might be “unbound” or “pipet-out”. Most scientists use “flow-through” here even though it is not technically correct. Whatever term you use – make sure to save this fraction for analysis.

Table 1. Example buffers for antibody purification with Proteins A, G, and L.

Wash

1. Remove the tube from the magnetic field and gently pipet in wash buffer. Close and gently invert the tube a few times to wash the beads.
2. Expose the tube to the magnetic field, pipet out the remaining liquid and save for further analysis.
3. Repeat this wash step 2-3 more times and save the liquid each time for further analysis.

Elute

1. Remove the tube from the magnetic field and pipet in the elution buffer. Close and gently invert the tube a few times.
2. Expose the tube to the magnetic field and pipet out the elution with your antibody. Save this for further analysis and downstream uses.
3. Repeat the elution step 2-3 times to ensure that you have eluted all of your antibodies off of the beads.

Neutralization

• Add neutralization buffer to your eluted sample to bring the pH to a more neutral value – typically pH 6 to 8.

Analyzing your Purification

You’ve (hopefully) purified your antibody! Now you’ll want to analyze the purification to see how it went.

1. Run an SDS-PAGE gel using the load, flow-through, washes, elutions, and neutralization samples.
2. Quantify your neutralized elution sample by measuring the absorbance of the solution at 280 nm wavelength of light, or by doing a Bradford assay.

The SDS-PAGE gel will give you a quantitative sense of how the purification went and may help answer some of the following questions (Figure 2):

• Did you effectively capture your antibody on the magnetic Protein A, G, or L agarose beads?
• Should you have used more or less magnetic agarose beads?
• Should you use a more, or less, stringent wash step?

Figure 2. Example of a hypothetical SDS-PAGE gel for antibody purification with Protein A, G, or L magnetic agarose beads. The antibody (purple band) will elute from the column and be in the neutralized solution whereas the other proteins will separate in the flow-through and wash fractions.

Quantifying your antibody will let you know what its concentration is and the total yield of the purification. Remember, protein purification is often an iterative process. If you purified any antibody – that is a success! Use the SDS-PAGE gel as a guide to show you how to optimize the purification if you do it again in the future. Use the quantification (concentration and yield) to benchmark whether future changes improve, reduce, or have no impact on the amount of antibody purified.

Additional References and Protocols

Protein A Protocol

Protein G Protocol

Protein A and Protein G Troubleshooting Guide

Affinity Comparison of Protein A and Protein G for Various Antibody Classes

Step-by-Step Protocol for Antibody Purification with regular Protein A, Protein G, and Protein L Agarose Beads

Regular vs Magnetic Agarose Beads: Key Differences and Best Uses

References

Imura, Y., Tagawa, T., Miyamoto, Y., Nonoyama, S., Sumichika, H., Fujino, Y., Yamanouchi, M., & Miki, H. (2021). Washing with alkaline solutions in protein A purification improves physicochemical properties of monoclonal antibodies. Scientific reports, 11(1), 1827. https://doi.org/10.1038/s41598-021-81366-6

Liu, H. F., Ma, J., Winter, C., & Bayer, R. (2010). Recovery and purification process development for monoclonal antibody production. mAbs, 2(5), 480–499. https://doi.org/10.4161/mabs.2.5.12645

Sheng, S., & Kong, F. (2012). Separation of antigens and antibodies by immunoaffinity chromatography. Pharmaceutical biology, 50(8), 1038–1044. https://doi.org/10.3109/13880209.2011.653493

Zarrineh, M., Mashhadi, I. S., Farhadpour, M., & Ghassempour, A. (2020). Mechanism of antibodies purification by protein A. Analytical biochemistry, 609, 113909. https://doi.org/10.1016/j.ab.2020.113909