Have you ever wondered whether you should be using regular agarose beads or magnetic agarose beads for your protein purification or experiment? If so, this article is for you!
Regular and magnetic agarose beads are both used for protein purification and immunoprecipitation. Regular beads are used for the large-scale purification of a few proteins at a time; whereas, magnetic beads enable small-scale purifications of several proteins in parallel.
From a big picture perspective, regular and magnetic agarose beads perform very similar functions, but how they go about doing those functions is slightly different from one another. In this article we’ll talk about the difference between regular and magnetic agarose beads, and why one can be more convenient than the other for certain applications.
Article Table of Contents
What are the Differences Between Regular vs Magnetic Agarose Beads in Protein Purification?
When do you use regular agarose beads?
When are magnetic agarose beads best to use?
What are the Differences Between Regular vs Magnetic Agarose Beads in Protein Purification?
Affinity purification of proteins and antibodies can be done with either regular agarose beads or magnetic agarose beads. There are a few slight differences in doing the purification with each type of bead, however.
Regular agarose beads:
With regular agarose beads, you allow the resin to settle to the bottom of a plastic column and use gravity to flow liquid through the beads. When you’re done washing contaminating proteins off of your column you can add an elution buffer, and your protein of interest will flow out the bottom of the column (Figure 1).
Figure 1. Regular agarose beads are packed into a plastic column. The protein of interest (green square) is bound to the tagged agarose bead. Contaminating proteins (pink and orange) are washed through, then an elution buffer is added to elute the protein of interest (little green squares).
Magnetic agarose beads:
Purifications with magnetic agarose beads are usually performed at a smaller scale, so the purification commonly happens in a tube instead of a plastic column. A magnetic force is applied to the tube and liquid is removed from the tube with a pipette. When you’re done washing contaminating proteins off of the beads you’ll add your elution buffer, apply the magnetic force, and then pipette out your protein of interest (Figure 2).
Figure 2. Magnetic agarose beads are loaded into a tube and exposed to a magnet. Contaminating proteins are pipetted out, then an elution buffer is added and the protein of interest is pipetted out.
So that’s the quick overview of the differences between using regular and magnetic agarose beads. In general, any application regular agarose beads can do, magnetic agarose beads can do as well, and vice versa. Yet, there are many scenarios where it is much easier to use one type of bead or the other. In the next few sections, we’ll talk about a few of these scenarios.
When do you use regular agarose beads?
Regular agarose beads are frequently the go-to choice when purifying one to a few proteins, and when purifying proteins at larger scale.
Purifying a lot of protein usually means that you have a large volume of input material, such as lysed cells or media with secreted proteins. When using regular agarose beads there is, practically speaking, really no upper limit to the input volume that you can use. You can just keep pouring additional material over the preformed agarose-bead column (Figure 1). Your protein of interest will keep binding to the agarose beads until you’ve saturated all of the ligand binding sites on the beads. The binding capacity of the beads you’re using will help you estimate how much agarose resin you need for your purification.
The downside of agarose beads, relative to magnetic beads, is that it is a little more tedious to purify more than one protein at a time. When purifying proteins by column, I usually try to limit myself to four or less to make sure that I can keep track of all of the samples and that I don’t let any of the columns run dry (run out of liquid).
As we’ll discuss next, magnetic agarose beads are really convenient for purifying different proteins or different conditions in parallel at the same time. If you have roughly 5 to 20 different purifications to do, it may be worth performing them at a smaller scale with magnetic agarose beads. If you need more of any of those proteins, you can always scale up and purify one, or a few winners, from your small-scale screen with regular agarose beads.
Table 1. GoldBio agarose beads for affinity purification.
|
Regular Beads |
Tag / Feature |
|
Nickel NTA |
His-tag |
|
Glutathione |
GST-tag |
|
Streptavidin |
Biotinylated Protein |
|
Protein A |
Antibodies – Heavy Chain |
|
Protein G |
Antibodies – Heavy Chain |
|
Protein L |
Antibodies – Light Chain |
When are magnetic agarose beads best to use?
Generally magnetic agarose beads are preferred when doing small-scale purifications and when doing many (~ 5 to 20) purifications in parallel.
Table 2. GoldBio 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 |
First off, why would you want to do so many purifications at the same time? Let’s say you had a bunch of variants of a single protein. Maybe these proteins have different domain boundaries or are point mutants of the wild-type protein (Figure 3). By screening these constructs with a small-scale purification first, you will save time and resources by quickly knowing which one(s) to move forward with for large-scale purifications.
Figure 3. For
a hypothetical protein (purple) with domains A, B, and C, one might want to
purify a variety of domain boundaries (left), and point mutations (right,
orange asterisks). Parallel purification using magnetic agarose beads can
streamline the process of purifying several slightly different proteins.
Magnetic agarose beads are more useful for small-scale purifications because of the tube size used for the purification (Figure 2). If using a standard 1.7 mL tube, the most you can use is a little less than that volume for the input material because a little bit of the volume will be occupied by the magnetic beads themselves. Of course, you can perform a series of load-and-rinse steps, but if you’re doing too many of those it probably would have been quicker and less of a hassle to just use regular agarose beads and the gravity-powered column. You can use a larger tube (15 mL or 50 mL) as well, provided you have the setup for applying a magnetic force to these tubes.
The racks used to apply a magnetic force to the tubes usually have slots for several (~ 8 to 24) tubes to be processed at the same time (Figure 4). This, along with the small volumes, is what makes it feasible to purify several different proteins or conditions in parallel. During the wash step, for example, you would put all of the tubes in the rack. After the beads are drawn towards the side of the tube with the magnet, then you would remove the liquid from each tube one at a time, then add wash buffer back into each tube. While it will take more time to purify more samples, you definitely save time compared to if you purified the samples serially.
Figure 4. Magnetic racks hold multiple tubes at the same time enabling rapid processing of multiple samples in parallel.
So those are main differences between regular and magnetic
agarose beads. Which type you use for a given purification will mainly be
decided by how many purifications you’re doing at one time, and the scale of
those purifications. Remember, these beads can be used synergistically by doing
many small-scale purifications with magnetic agarose beads, then scaling up the
promising proteins with regular agarose beads.
