Does it ever feel like the hardest part of a project is just getting started on the dang thing? For lots of projects, it feels like there are a hundred slightly different ways that you could accomplish your goals. And when you don’t have much prior experience in the area, it can feel really difficult knowing where to start.
Ion exchange chromatography is kind of like that. There are many different ways you could use ion exchange chromatography to purify your protein, and it is sometimes paralyzing to pick one to start with.
To purify your protein using ion exchange chromatography (IEX), you’ll load your protein onto a column with beads that are the opposite charge as your protein. After washing away contaminating proteins, you’ll elute your protein by increasing the salt or changing the pH of your buffer.
In ion exchange chromatography, you elute your protein with a step elution, a gradient elution, or a combination of the two. The best elution type to use will depend on the protein you are purifying, it’s intended purpose, and whether you are employing any other purification steps.
In this article we’ll describe the difference between step and gradient elution, cover a go-to protocol to use on proteins you haven’t purified before, and cover how you can optimize this generic protocol for difficult proteins or specific purposes.
Article Table of Contents:
Ion Exchange Purification Protocols
Ion Exchange Purification Protocols
Like affinity tag purifications, ion exchange protocols include discrete load, wash, and elute steps (Figure 1). For the load step you will want your salt concentration to be low enough that your protein will stick to the column. If you’re using NaCl, typically somewhere between 50 and 150mM is a good concentration for loading your protein onto the column.
Figure 1. Ion-exchange chromatography has distinct load (left), wash (middle), and elute (right) steps.
Beware, that some proteins have low solubility at low salt concentrations and will aggregate. For this reason, you usually want to lower the salt enough that it will bind to the column, but no more than that. When working with a new protein it may take some trial and error to find this happy medium between column binding and aggregation. Alternatively, if you don’t lower the salt enough, your protein of interest won’t bind to the column and will come out during the load and wash steps.
Next, you’ll want to wash the column to remove any contaminating proteins. Typically, this is done with the same buffer you loaded your protein onto the column with.
Last, you’ll elute your protein off of the column. There are a few options here for how you do this. First off, you could use a step elution or a linear gradient elution (Figure 2).
Figure 2. Shows the difference between salt addition in step elution (left) versus a linear gradient elution (right). Step elution immediately raises the salt while gradient elution increases salt gradually.
The advantage of a step elution is that it is relatively fast. The disadvantage of using a step elution is that you may have more contamination with other proteins compared to a gradient elution.
During a step elution, you immediately raise the salt to a high enough concentration that it disrupts the electrostatic interaction between the column beads and your protein of interest (Figure 2, left), like 500mM or 1,000mM NaCl, for example.
For a gradient elution, you gradually increase the salt concentration (Figure 2, right), which allows different proteins to elute at distinct times. This is important, because if there are other contaminating proteins in your sample, a gradient elution may do a better job of separating your protein of interest from these contaminants.
A standard protocol that I use is to increase the salt concentration from the loading buffer to 1,000mM NaCl over twenty column volumes (CVs). A column volume is just as it says – how much volume your column holds. If you’re working with a 5 milliliter (mL) column, then 20 CVs is 100 mL (20 x 5 mL). So, for example, if your loading buffer is 100mM NaCl, then you can ramp the salt from 100mM NaCl to 1,000mM NaCl over 100 mL of volume. That may be a lot to follow – but check out Figure 3, and it will all make sense.
Figure 3. Example of a typical ion exchange program with a hypothetical 5 mL column. This is a good go-to protocol when working with new proteins that you haven’t purified with ion exchange chromatography before.
The more you know about your protein’s behavior, the more you can optimize around it. For example, if from your first purification you know that your protein elutes at 300mM NaCl, you may deviate from the previously described purification protocol and instead elute your protein using a shallower linear gradient (Figure 4).
Figure 4. Example of a shallow ion exchange elution with a hypothetical 5 mL column. This is a good protocol to run for a protein that elutes in the 100 to 400mM NaCl range and needs to be separated from contaminating proteins that also elute in this range.
In this hypothetical example you have 20 mL of protein sample to load onto the column, you wash for 10 mL (2 column volumes), just as before with the previous protocol. Then, the difference with this one is that you do a gradient elution from 100mM to 400mM NaCl over 100 mL (20 column volumes). This is a good purification procedure to use on a protein that elutes between 100mM and 400mM NaCl, and needs a shallow gradient to separate a contaminant or different conformations, for example.
Elutions don’t have to be purely step or gradient – you can incorporate both into more complex protocols. After you purify a protein with ion exchange once or twice, you may be able to come up with an advanced protocol that combines the time and reagent-saving advantages of step elution with the improved purification resolution of gradient elution (Figure 5).
Figure 5. Example of an ion exchange purification protocol that features both gradient and step elution components. This particular protocol is used for the purification of Xrn1, an RNA exonuclease (Pellegrini et al, 2008).
A standard ion exchange purification protocol, such as the one described in Figure 3, will probably work just fine for most proteins that you purify. However, the general guidelines we covered in this article will help you optimize your purification protocol for the few proteins that exhibit peculiar behavior, need a little extra purification, and when you’re separating very similar proteins from one another.
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