Size matters! Like on roller coasters, for example. Everyone has seen the sign that says, “you must be this tall to ride the roller coaster.”
Now, you’re probably wondering why I’m talking about roller coaster rules in an article about protein purification. It’s because this is such a good analogy for protein purification using size exclusion chromatography.
Size exclusion chromatography purifies proteins according to their size. Larger proteins elute first whereas smaller proteins meander through bead pores and elute later. Size exclusion is useful for isolating different proteins and separating the same protein in different oligomeric states.
Like roller coasters, size exclusion chromatography has its own size selection process for purifying proteins. In essence, there are a lot of tiny beads with signs that say, “you must be smaller than this to enter the bead.” By combining beads with a variety of pore sizes, proteins are separated based on their size. We’ll explain more about how exactly this process works in this article.
Article Table of Contents:
Plain vs Crosslinked Agarose Beads
Size Exclusion Chromatography Purification Protocol
Important Uses for Size Exclusion Chromatography
Separating Different Molecules by Size
Separating the Same Protein by Oligomeric State
Size Exclusion Beads
Agarose beads are a popular choice for size exclusion chromatography (SEC) because they are good at purifying larger biomolecules such as proteins. Additionally, agarose beads are typically inert – they usually don’t bind to proteins, making them ideal material for protein purification.
Think of it this way; affinity and ion exchange chromatography steps are like Velcro in that we want our protein of interest to stick to the column and then later we remove the proteins from the column. Size exclusion chromatography is more like a mouse maze where the protein needs to navigate through the column. And we don’t want the protein to get stuck on the beads as it meanders through them.
When choosing which type of agarose beads to use there are a few important considerations, such as:
- what percentage of beads to use?
- plain vs. crosslinked agarose beads?
Agarose Bead Percentage
A higher percentage of agarose beads is useful for purifying smaller proteins, and a lower percentage of agarose beads is used to purify larger proteins. As an example, let’s compare 4% and 6% agarose beads. While every protein is a little different, a general rule is that you would want to use 4% agarose beads for purifying a protein larger than 30 kilodaltons and use 6% agarose beads for purifying a protein smaller than 10 kilodaltons.
Plain vs Crosslinked Agarose Beads
Crosslinking is a modification that covalently links different beads together, whereas plain agarose beads lack these crosslinks and are floating around on their own. While both types of beads are used for size exclusion chromatography, there are important differences between these two variants.
To put it simply, crosslinked agarose beads are sturdier and can withstand harsher experimental conditions. For size exclusion chromatography, three relevant examples of harsh experimental conditions are the buffer, temperature, and pressure.
Crosslinked beads can withstand a wider pH range during purification and cleaning of the column (Ó'Fágáin et al, 2017). For example, it’s helpful to clean your SEC resin every few runs to get rid of any sticky protein that remains. Sodium hydroxide, a strong base, is a common cleaning reagent that will remove most sticky residual protein material. Sodium hydroxide cleans crosslinked beads very well, and the beads are then used again for additional purifications, effectively extending the life of the beads. However, plain agarose beads are too delicate, and sodium hydroxide irreparably damages them.
For some applications, people also want to sterilize their agarose beads, and this is commonly done by autoclaving them. Crosslinked beads withstand the high temperatures of autoclaving, but you wouldn’t want to do this to plain agarose beads as it would destroy them.
Plain agarose beads work just fine for gravity columns. This is when you pour your resin into a plastic column and allow gravity to “pull” the liquid through the column. There are also liquid chromatography systems such as FPLC and HPLC that apply additional pressure to run the columns faster, and often with better separation between biomolecules. Crosslinked agarose beads are needed when additional pressure is being used because plain agarose beads crack under pressure (it’s a joke … but also it’s true).
So which type of beads should you use for size exclusion chromatography? If you use gravity columns and perform SEC infrequently, it probably makes the most sense to buy the plain agarose beads and discard them after each use. However, if you frequently purify proteins with SEC, and especially if you use higher pressure applications or want to autoclave your beads, then you’ll definitely want the crosslinked or HTC agarose beads. Although the crosslinked beads cost a little more up front, proper cleaning and reuse of the crosslinked beads will be the more cost-effective route for frequent users over the long run.
Figure 1. Agarose beads have different pore sizes. Small proteins fit into agarose beads with small pores (left), whereas small and medium proteins fit into agarose beads with medium pores (middle right). The more beads a protein fits into the slower its journey through the agarose resin will be (Figure 2).
In this way proteins are separated from each other according to their size (Figure 2). As the proteins come out of the bottom of the column, you will collect elution fractions.
By measuring the protein concentration of each fraction, you can then generate a chromatogram, which is a plot that shows you how much protein you have versus the time or volume that your SEC purification has been running. Figures 3 through 7 show hypothetical examples of size exclusion chromatograms. Peaks refer to the elution fractions that have a high concentration of protein, and when we are trying to separate different types of proteins, we are trying to optimize the separation of these protein peaks.
Note that there are automated systems, such as FPLCs and HPLCs, that will automatically generate chromatograms and fractionate the SEC elution. However, if you are running a gravity column, you will need to perform these steps by hand.
Figure 2. Protein mixture purified by size exclusion chromatography. Larger proteins elute faster, and smaller proteins elute slower. Since the purification process starts as soon as the protein mixture is added to the column, a relatively small protein volume can be added each time.
SEC is a fundamentally distinct type of purification compared to affinity and ion exchange chromatography. In affinity and ion exchange chromatography there are discrete load, wash, and elute steps. Put another way, you can load as much volume as you want with these types of purifications because your protein will remain bound to the resin beads until you elute them.
As we described above, size exclusion chromatography works a little bit differently. As soon as you start applying your protein solution to the resin, the proteins will start traveling through the column.
Since protein separation starts as soon as you apply the sample to the column, there’s a limited protein sample volume you want to apply for each SEC purification run. Overloading the column will result in poor separation because large proteins loaded later will overlap with medium proteins loaded earlier, for example (Figure 3).
Check the manufacturer’s recommendation for the beads that you are using, but a general guideline is to load around 1 to 5% of the column volume per run. So, if the column volume is 100 mL, for example, load at most 5 mL of protein sample.
Figure 3. Limiting the load volume to less than 5% of the column bead separates chromatogram peaks containing different proteins (purple, pink, and blue circles) (left). Overloading the column results in peaks blurring together and poorer separation of different proteins (right).
Buffer Guidelines
Compared to other purification steps, the types of buffers used for size exclusion chromatography are pretty flexible. Crosslinked beads work well from pH 3 to 11, which is a pretty broad pH range that should work for most types of proteins (Ó'Fágáin et al, 2017). There is no strict rule on how much salt to include or avoid, but you’ll want to include enough salt so that your protein won’t stick to the agarose beads. This will vary depending on the specific protein, but in general 0.15 – 0.2 molar NaCl is usually sufficient.
Typically, the buffer you use in SEC will be dictated by your intended downstream use. Speaking of which, size exclusion is a convenient opportunity to exchange buffers into exactly what you need your protein to be in for the types of experiments you are going to conduct. It is also a nice cleanup step to get rid of any small molecules that might be present in your sample from upstream purification steps, such as imidazole, ATP, or biotin for example.
If your first choice of buffer does not sufficiently purify your protein by SEC, you can always go back and reoptimize buffer conditions such as pH and salt concentration. However, in most cases keeping it simple in terms of SEC buffer composition works well.
Important Uses for Size Exclusion Chromatography
Size exclusion chromatography is often used as the last step in a multistep protein purification process. For example, you might first do an affinity purification step, then ion exchange chromatography, and lastly size exclusion. In this case, SEC is sometimes a polishing step in that there isn’t a whole lot of purification left to do if the first two steps went well.
However, there are a few important uses for size exclusion chromatography including:
- separating different molecules by size.
- separating the same protein based on oligomeric state.
- Estimating protein size.
Separating Different Molecules by Size
Size exclusion chromatography separates different molecules by size (Figure 4). For your purification purposes this may be useful in isolating your protein of interest from contaminating proteins. It may also work to separate your protein of interest from other side products from earlier purification steps, such as a cleaved affinity tag or the protease you used to cleave the affinity tag, for example. Lastly, SEC is great at separating proteins from much smaller molecules that you may have used in previous purification steps such as imidazole, ATP, or biotin.
Figure 4. Larger proteins elute earlier, and smaller
proteins elute later in size exclusion chromatography.
Separating the Same Protein by Oligomeric State
SEC is also useful for separating your protein of interest from itself. This isn’t a philosophical statement. Actually, it is quite practical. Purified proteins often exist in equilibrium between different oligomeric states, such as between a monomer and a dimer. If the interconversion between those two states is sufficiently slow, you will see evidence of this equilibrium in your SEC chromatogram (Figure 5).
Figure 5. For proteins in a monomer-dimer equilibrium (left) those species are often resolved via size exclusion chromatography (right).
An important question here is whether the interconversion is
sufficiently slow that you have effectively separated the monomer and dimer, or
whether both peaks contain protein that are still in the monomer-dimer
equilibrium. One way to distinguish between these two possibilities is to take
the dimer fraction and rerun it through SEC (Figure 6). If you’ve separated the
two species, you will see just the dimer peak on this second run. However, if
the interconversion is sufficiently fast, you’ll still see both species.
If you just see the dimer peak on the 2nd SEC run, this means you’ve effectively separated the two species. In this case, you likely want to keep the monomer and dimer peaks separate for downstream purposes because there may be important differences in your experiments depending on if you’re working with the dimer or the monomer.
However, if you see dimer and monomer peaks again on the 2nd SEC, this means that the interconversion is too fast to separate. So even though you’re seeing “dimer” and “monomer” peaks, the molecules in both of those fractions are still converting back and forth between monomer and dimer. In this case, you won’t be able to effectively separate these states with SEC, but you probably want to note that you’re observing this behavior and then combine both species together for whatever experiments you have planned with that protein.
Note that while I’ve used a monomer-dimer equilibrium in this section, the overall point still applies even if you’re dealing with a different type of oligomeric interconversion (monomer-trimer, dimer-tetramer, etc.).
Estimating Protein Size
Size exclusion chromatography can estimate the size of your protein. There are protein standards that you run on your SEC column with the same buffer and flow rate as your protein of interest. This can help you estimate the size of your protein (Figure 7).
Figure 7. By comparing your protein of interest (POI, magenta) to a set of protein standards (gray, molecular mass in kilodaltons) on SEC, you can estimate the size of your protein. For example, this protein elutes at a volume that corresponds to approximately 40 kilodaltons.
For example, the protein of interest (POI) in Figure 7 elutes in between the 50 and 25 kilodalton protein standards. It’s a little bit closer to the 50 kilodalton peak, so I would estimate the protein at roughly 40 kilodaltons. You can calculate your protein’s predicted molecular mass and compare it to this result. Suppose your protein is around 20 kilodaltons in mass, then this peak would probably indicate that your protein is in a dimer.
This method is a size estimation, and there are additional methods that can be hooked up downstream of SEC such as SEC-MALS (multi angle light scattering) if you need a more precise analysis of your protein’s size (Ogawa & Hirokawa, 2018).
Hopefully now you see why, like roller coasters, size matters for protein purification by size exclusion chromatography!
