What are Agarose Beads? A Deep Overview
by Simon Currie
Do you ever feel like you’re bouncing through life, squeezing through the never-ending to-do items on your list? If so, then you have a good idea of what it feels like to be a protein or nucleic acid traveling through a column of agarose beads.
Agarose beads are porous, spherical hydrogel particles made from seaweed polysaccharides. Agarose beads are a critical tool in biotechnology for purifying proteins and nucleic acids, performing pull-down or immunoprecipitation experiments, and more.
In this article, we’ll give a broad overview of agarose beads and what they’re used for. We have many other articles that go into the specific details that we’re broadly covering here, so look for those if you’re interested in learning more about a particular subject.
Article Table of Contents
Conjugating affinity ligands onto agarose beads
What are agarose beads used for?
What are agarose beads?
Agarose beads are small micro-scale beads made from agarose polysaccharides of seaweed. Agarose beads can have different properties, such as pore size and rigidity, based on how they’re formulated. Additionally, some agarose beads are decorated with affinity ligands so that they can be used to purify tagged-proteins and as a support for binding experiments.
Let’s get into some of the details about how agarose beads are made so we can explore where these different properties come from.
How are agarose beads made?
Have you ever made Jell-O®? Making agarose is essentially the same process. You dissolve the desired amount of agarose in liquid, heat it up, then let it cool down into the shape that you desire.
Heating agarose fully dissolves it in the liquid and detangles all of the individual polysaccharides. Then, as it cools back down the polysaccharides will re-intertwine, thereby forming a solid (Figure 1).
Figure 1. When agarose is heated up, individual polysaccharides disentangle and it is a free-flowing liquid (left). As agarose cools, such as in an agarose gel, the polysaccharides become entangled and form pores through which molecules travel (right).
This is the same process if you’re making an agarose gel for analyzing DNA molecules. After melting the agarose, you then add your stain of interest, and then pour it into the gel casting tray and add the gel comb.
Making agarose beads is a similar process conceptually, with a few additional complications. Before cooling, the hot agarose solution is added to a mixture of a hydrophobic liquid, emulsifier, and surfactant. This mixture is then constantly stirred as it cools to form agarose beads (Figure 2). For more details on this process, check out this article about how agarose beads are made.
Figure 2. Process for making agarose beads. Hot aqueous agarose solution and surfactant are added to a hydrophobic liquid and emulsifier. As the solution cools with continuous stirring the agarose beads will form.
This is a general procedure for making agarose beads, but there’s a few additional parameters that are important to be aware of when working with agarose beads:
· Agarose percentage
· Crosslinking
· Affinity ligands
Agarose percentage
This is the percent (weight/volume) of agarose that went into making the agarose solution. For agarose gels, this usually ranges from 0.5 – 2.0 %. Beads are a higher percentage agarose, around 4 – 6 %.
Agarose percentage mainly impacts two key features: pore size and rigidity.
The lower the agarose percentage, the larger the pore size will be. Intuitively, it makes sense that the more agarose polysaccharides you have in your solution, the more entangled they will be upon cooling, leading to smaller pore sizes. Smaller molecules will be able to pass through the smaller pore size, whereas larger molecules will not fit (Figure 3).
Figure 3. Lower percentage agarose will have larger pore sizes, so small and medium molecules will fit through their pores (left). Higher percentage agarose will have smaller pore sizes, so only small molecules will fit through the pores (right).
The higher the agarose percentage, the more rigid the gel or beads will be. Again, this makes intuitive sense that the more interactions that are occurring between different agarose polysaccharides, the stronger the resulting structure will be. You can think about this like a kid building a play bridge out of sticks. If there are just a couple of sticks holding up the bridge, it will be pretty flimsy. However, if the kid has really reinforced the bridge with a lot of sticks, there is a better chance that the bridge will stay standing.
Crosslinking agarose beads
Another parameter to be aware of for agarose beads is crosslinking. This is where an additional chemical is added that will link together different agarose polysaccharides. Similarly to agarose percentage, adding crosslinkers changes the pore size and rigidifies, or strengthens, the resulting beads (Figure 4).
Figure 4. Crosslinkers (green) make smaller pore sizes and make agarose beads more sturdy and rigid.
Let’s go back to the stick bridge analogy. Now instead of just using sticks to build the bridge, the child has used rope to bundle together multiple different sticks. This is like the crosslinker for agarose beads in that bracing together different sticks will make the bridge stronger and more resilient.
As a general rule, if you’re going to use agarose beads once and then discard them, then regular (non-crosslinked) beads will work fine.
However, if you plan on reusing your agarose beads, and/or autoclaving them, then you will definitely want to go with the stronger crosslinked beads.
Conjugating affinity ligands onto agarose beads
So far, we’ve been talking about plain, undecorated agarose beads, which have a few uses that we’ll cover in the next section.
Frequently, scientists work with agarose beads that have ligands added onto them. These ligands are conjugated onto the agarose beads using chemical reactions that you could learn more about in this article. There are a broad range of ligands that are added onto agarose beads, from small molecules to proteins and even antibodies.
Once added onto the agarose beads, you have a solid support that is used to either purify proteins that interact with the ligand, or to investigate molecular interactions.
What are agarose beads used for?
Now that we’ve covered some of the fine details about how agarose beads are made, let’s move on to what they’re used for.
Two main uses for agarose beads are for purifying protein or DNA molecules, and as a support system for interaction experiments.
Size exclusion chromatography
Let’s start with plain, unliganded agarose beads. These are used for size exclusion chromatography, which separates molecules based on their size. Small molecules will travel through lots of the pores in agarose beads, which makes their effective path length through the column longer. Large molecules won’t fit into as many pores, and so their effective path length through the column will be shorter (Figure 5). For more details on how size exclusion chromatography works, see this article.
Figure 5. Size exclusion chromatography separates proteins by size. Large proteins (purple) will be excluded from agarose beads and will have a shorter path through the column. Small proteins (blue) will travel through many beads and have a longer path length. Medium proteins will travel through some beads with larger pores and have an intermediate path length.
This is an application where the percentage of agarose will make a difference. Higher agarose percentage will result in smaller pore sizes, which will be good for separating smaller proteins or nucleic acids. Lower agarose percentage will result in larger pore sizes which is better at separating larger molecules.
Affinity chromatography
Agarose beads are frequently used for affinity chromatography when they have ligands conjugated to them. A variety of ligands are used, which bind to different protein tags. For example, his-tags bind to nickel agarose beads, GST-tags bind to glutathione agarose, and biotinylated molecules or strep-tags bind to streptavidin beads.
Each of these types of beads purify proteins in a similar fashion with bind, wash, and elute steps. Your tagged protein of interest will bind to the column while other proteins flow through. You’ll wash the column to get rid of as many contaminating proteins as possible, then elute the tagged proteins by adding a buffer component that interferes with the ligand-tag interaction (Figure 6).
Figure 6. Tagged proteins bind to liganded agarose beads while contaminating proteins flow-through and are washed out of the column (left and middle columns). Lastly, the tagged protein is eluted from the column by adding a buffer component that competes with the ligand-tag interaction.
For example, imidazole is used to elute his-tagged proteins from nickel beads and glutathione is used to elute GST-tagged proteins from glutathione beads. Eluting biotinylated or strep-tagged molecules from streptavidin beads is a little more complicated, but using excess biotin is one of several ways that can work.
Binding experiments
Binding experiments use the ligand-tag interaction to isolate your tagged protein of interest. By adding in other molecules, or a complex biochemical mixture like cell lysate, you can see which molecules bind to your protein of interest.
GST pulldowns or immunoprecipitation are the most common version of this type of experiment, but you can perform analogous isolation experiments with other protein tags including his-tags and strep-tags (Figure 7).
Figure 7. Nickel agarose (left), streptavidin agarose (middle), and glutathione agarose (right), are used to detect interactions with his-tagged, strep-tagged, and GST-tagged target proteins, respectively.
That’s the basics of agarose beads, from how they’re made to how they’re used. If you’re interested in learning more about agarose beads and their applications, we have many links to additional articles sprinkled throughout this article, and below. Also, GoldBio has a lot of great agarose bead products to kick start your research, so check those out as well.
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