If you need to purify a protein for your research, but you’re not sure exactly which purification techniques to use, this is just the article for you!

There are lots of different ways to purify a protein, and many purification protocols combine a few different techniques to generate pure protein for various research applications. But how do you know which are the best purification techniques to use for your protein?

Choosing the right protein purification techniques involves three steps. First, build off previous successful purifications. Second, use your knowledge about the protein and its intended purpose for initial design. Third, adjust and optimize after you’ve purified the protein a few times.

In this article we’ll briefly overview the different types of protein purification techniques and then discuss the strengths of each type and how to combine the individual techniques for an overall purification scheme. We also have a lot of articles that go into more detail on each individual purification technique, and you’ll find links to those throughout this article.

Article Table of Contents:

Types of Protein Purification

Affinity Chromatography

Ion-Exchange Chromatography

Hydrophobic-Interaction Chromatography

Size-Exclusion Chromatography

Combining Protein Purification Techniques for an Overall Protocol

Related Products

References



Types of Protein Purification

First, let’s briefly look at the main types of protein purification techniques, which include:

  • Affinity
  • Ion exchange
  • Hydrophobic interaction
  • Size exclusion



Affinity

Affinity chromatography makes use of a small peptide tag or solubility protein domain tag to bind to specific resins (Figure 1). For example, a common affinity tag is a histidine tag which uses repeats of histidine residues, usually 6 to 10 consecutive histidines, to bind to nickel-coated agarose beads. Similar transition metals such as cobalt, zinc, and copper can also be used in lieu of nickel.

Overview of affinity protein purification

Figure 1. The target his-tagged proteins (purple) bind to nickel agarose beads. Nontarget proteins (orange and red) flow through the column during the bind and wash steps. His-tagged proteins (purple) are then eluted from the column.


Affinity purifications have distinct load, wash, and elution steps (Figure 1). During the binding step the protein is loaded onto the beads. Nontarget proteins are then washed off the column. Lastly, the target protein is eluted off the column by disrupting the interaction between the affinity tag and agarose beads.

If your protein construct has an affinity tag, or if you plan to clone a new plasmid with an affinity tag, then affinity chromatography is generally used first in an overall purification protocol. Pure protein can be obtained for most protein constructs by optimizing expression conditions, purification buffers, and the amount of affinity resin used during this step.



Ion Exchange

Ion-exchange uses a protein’s charge to purify it by binding to a resin of opposite charge (Figure 2). It’s great at purifying native proteins that lack affinity tags, as well as separating very similar proteins that differ slightly due to:

  • Oligomeric state
  • Conformational state
  • Posttranslational modification

If your protein lacks an affinity tag, you will probably want to use an ion-exchange purification step, or two, to isolate your protein. Similarly, if you’re having trouble separating any of the subtle differences mentioned above, ion-exchange is definitely the way to go to resolve these similar species.

Ion exchange chromatography illustration

Figure 2. Ion-exchange chromatography also has distinct load (left), wash (middle), and elute (right) steps.



Conversely, if you want to keep all of the conformational states of your protein – say because you want to visualize all of them by cryo-EM – then you wouldn’t want to use ion-exchange to purify your protein. We’ll discuss this issue in a little more depth in the section about combining different purification techniques at the end of the article.

Also, you usually need to use a buffer with relatively low salt concentrations to bind your protein to the ion-exchange resin. If your protein is unstable in low salt conditions, ion exchange might not be the right purification technique for that protein.

Table 1 summarizes this information in an easy guide that shows when ion exchange chromatography for protein purification is a good option to use, and when it would not be ideal.


Table 1. Summary of when ion exchange purification is recommended.

Situation

Ion Exchange Recommended?

Purifying native proteins

✔️ Yes

Purifying proteins with no affinity tag

✔️ Yes

Protein species differ slightly (oligomeric states, conformational states, posttranslational modifications)

✔️ Yes

Need high-resolution separation of similar proteins

✔️ Yes

Protein stable in low salt concentrations

✔️ Yes

Need to retain all protein conformational states for study

❌ No

Protein unstable in low salt conditions

❌ No


Hydrophobic Interaction

In a way, you can think about hydrophobic interaction as a complementary technique to ion exchange. Instead of using a protein’s electrostatic properties to bind it to the column, you’re using the protein’s hydrophobicity to do so. In lieu of using low salt, like in ion exchange, you use high salt in the binding buffer to promote hydrophobic interactions between the resin and your protein. Then the salt is lowered until your protein releases from the column.

Since you’re using relatively high salt in the loading step, this can be a good technique to use for proteins that are unstable in low salt and are not a good fit for ion exchange. On the other hand, if your protein is not stable in high salt conditions, you want to avoid hydrophobic interaction chromatography and probably do ion exchange instead.



Size Exclusion

Size exclusion chromatography separates proteins according to their size. You can think about this technique like a boat race. Small, medium, and large proteins are racing through agarose beads with different pore sizes (Figure 3). The small proteins are drawn into all of the agarose beads and will take longer to elute, whereas larger proteins will pass around the agarose beads and elute earlier (Figure 4).

Illustration of an agarose bead and pore for protein purification

Figure 3. 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 (right). The more beads a protein fits into, the slower its journey through the agarose resin (Figure 4).



This technique is really useful for separating proteins by size and can even separate the same protein based on the oligomeric state. See this article for more details about size exclusion chromatography and its uses.

illustration of size exclusion chromatography for protein purification

Figure 4. 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.



Combining Individual Techniques for an Overall Purification Protocol

Ok, so now that we’ve briefly covered the main types of protein purification, how do you choose which to combine for a successful protocol? In essence how you craft a purification protocol will depend on the protein you’re purifying, its biochemical properties, and what purpose(s) you are purifying the protein for.

A typical purification scheme that I frequently use is to first do an affinity purification, then ion exchange, and lastly size exclusion (Figure 5). If I’m cleaving off the affinity tag, then I’ll usually do that after the affinity purification and re-run the affinity column again to deplete the cleaved tag and any un-cleaved fusion protein. This overall purification protocol works well for most proteins and for most intended purposes, but there are certainly reasons to deviate from this standard scheme.

protein purification protocol map

Figure 5. A standard protein purification protocol that I frequently use. The additional affinity step to the bottom (dashed lines) is only used if removing the affinity tag during the purification.

In general, if you’re purifying protein for a qualitative purpose – something where a binary yes/no answer will suffice – then you can probably get away with performing fewer purification steps, perhaps even just a single affinity purification. In contrast, if you’re using your protein for quantitative analyses or for structural biology experiments, then purity is usually very important, and you’ll likely want to keep at least these 3 standard purification steps in your protocol.

Even within structural biology purposes, purification needs can vary. In X-ray crystallography, you first form a protein crystal and pure protein is very important for the formation and quality of such crystals. Additionally, crystallography is good at getting a very precise picture of your protein in exactly one state or conformation. For that reason, it is often important to use ion exchange chromatography to confirm that there is only a single conformation, or to separate different conformations if they exist.

In cryo-EM, you can image all of a protein’s different conformations. So, you usually don’t want to purify a protein intended for cryo-EM with ion exchange chromatography because then you would be losing useful information in the process.

These are a few illustrative examples – but the general point is that you should think critically about your protein, it’s properties, and what you want to use it for when designing a purification scheme.

This is the kind of process where you most likely won’t get it perfect on the first try – so don’t worry about that! Instead, devise your best guess at what will work well for your protein. It’s important here to rely on any successful previous purifications in the literature and build off of those. After you’ve purified your protein a few times – or at least tried – you’ll have a lot more information to work with to optimize your purification protocol.

So that’s the basics about how to design a purification protocol. If you’re interested in reading more about any of these individual techniques, please use the links throughout to connect to our many articles on these subjects. Also, if you’re ready to start purifying protein, see the links below to many of our excellent protein purification related products.