Agarose beads are a foundational tool used for various protein purification techniques. Most frequently, agarose beads are purchased through a vendor such as GoldBio. However, you may be curious as to how agarose beads are made, or you may be interested in optimizing a particular kind of agarose bead for a custom-fit purpose. If so, this is the article for you.
Agarose beads are formed by melting an agarose solid into a hydrophobic liquid and emulsifier. Surfactant is added, and the solution is stirred constantly as it’s cooled to form the agarose beads. The beads are then washed, selected for particular sizes, and crosslinked if desired.
In this article we’ll cover the basic steps about how agarose beads are made and discuss crosslinking of agarose beads.
Making Agarose Beads
Agarose is extracted from seaweed and contains long polysaccharide (carbohydrate) polymers consisting of repeating units of 1,3-linked b-D galactose and 1,4-linked 3,6 anhydro-a-L-galactose (Figure 1). In practice, agarose or agar is purchased as a solid powder (see links below), weighed out, and melted in an aqueous solution such as water. The heat disrupts interactions between different polysaccharide polymers, resulting in the agarose now being in the liquid phase.
Figure 1. Agarose
monomer.
Agarose is a polymer made up of the monomer subunits (1,3-linked b-D galactose and 1,4-linked 3,6 anhydro-a-L-galactose) shown inside the bracket. The
bracket and letter “n” signify that agarose is made up of thousands of these
molecules strung together.
If you’ve ever made an agarose gel for separating or analyzing DNA then you know that so far, the steps have been essentially same for making agarose beads. In the case of the agarose gel, you would pour the heated agarose liquid into a container in the shape of your desired gel, and let it cool down. During the cooling process polysaccharides become entangled resulting in the formation of a solid agarose gel.
For agarose beads, however, the cooling process is a little more complicated. The heated aqueous agarose solution is added to a hydrophobic liquid and emulsifier, and this solution is constantly stirred. Examples of hydrophobic liquids used to make agarose beads include toluene, mineral oil, and liquid paraffin and petroleum (Cheng et al, 2010; Li et al, 2015; Zhou et al, 2007). Surfactant is also added, and the solution is continuously stirred as it cools down (Figure 2)(Nweke et al, 2017).
Each of these parameters – the type and amount of hydrophobic liquid, emulsifier, surfactant, and stirring speed – are important in determining the size of agarose beads and the consistency of size from bead to bead in each batch. For instance, stirring and cooling rates are important parameters in determining bead size distribution, bead porosity, and pore size distribution (Ioannidis, 2009). Typically, agarose beads range from 20 – 300mM in diameter, and you can intentionally bias the reaction towards the longer or shorter end of this range by adjusting the stirring and cooling rates (Andersson, 2014). For an example of why bead size matters when purifying proteins – see this article.
Figure 2. Making
agarose beads.
A hot aqueous agarose solution is added to hydrophobic
liquid and emulsifier with constant stirring (left). Surfactant is added and
then the solution is slowly cooled to form agarose beads (right).
After the cooling steps, the agarose beads will have formed. Then, the beads are washed to remove the reaction components that are no longer needed and may not be desirable for exposing to biological samples, such as the hydrophobic liquid, emulsifier, and surfactant.
Then the beads are passed through a sieve and collected. You can think about this like straining spaghetti, except in this case it would be the pasta water that you want to keep. The sieve removes any excessively large beads whereas the appropriately sized beads pass through the sieve and are collected for further use (Figure 3). In this way, both the washing and sieving steps contribute to selecting the appropriate bead size by removing beads that are too small and too large, respectively.
Figure 3. Sieving
agarose beads.
Sieving agarose beads removes beads that are larger than the
desired size (top) while keeping beads that are the desired size (bottom).
To Crosslink or not to Crosslink?
At this point, the agarose beads are ready for the optional step of crosslinking. Crosslinking is adding chemical modifications that staple different agarose polymers together (Figure 4). But first let’s discuss why you might want, or not want, crosslinked beads.
Figure 4. Plain vs
crosslinked agarose.
Agarose polymers are represented by gray lines. In
plain beads, the agarose polymers are by themselves (left). In crosslinked
beads there are chemical links between the agarose strands that strengthen the
beads.
Agarose vs Sepharose Beads
Agarose beads are sometimes called by brand names such as “Sepharose” or “Superflow” by different companies. Fundamentally, these products are all just agarose beads. However, as we’ve discussed differences in how the beads are made and crosslinked lead to important differences in agarose bead size, properties, and sturdiness. It’s important to consider any specifications the company provides for their beads, and search for any existing head-to-head comparisons to determine what will work best for your experimental setup (Nweke et al, 2017).
Those are the basic steps of how agarose beads are made. We have a related article on how ligands are conjugated to agarose beads for affinity purification, and a ton of articles on the different types of agarose beads that GoldBio sells, so if you want to learn more about agarose beads dive down the rabbit hole with all of those articles!
