Understanding Salting In and Salting Out: Salt as a Protein Purification Tool

Salt is a major component in protein purification buffers, and optimizing this important parameter can make or break a purification step. But, did you know that salt itself can be a purification step?

Salting in is adding salt to a solution to enhance the solubility of a protein, whereas salting out is adding salt to precipitate a protein. These techniques leverage the differential solubility of distinct proteins in various salt conditions to partially purify proteins.

In this article we’ll discuss salting in and salting out, the importance of salt types on these processes, and how these processes are utilized in protein purification schemes.

What is Salting In and Salting Out?

Salting in is adding salt to a solution to make a protein more soluble. This is like a Goldilocks-type situation where for most proteins there is a “just right” amount of salt that maximizes a protein’s solubility. Once you go past that threshold, then by adding more salt, you will start salting out the protein and causing it to precipitate.

By the way, in case you’re not familiar with the classic English nursery story, Goldilocks and the Three Bears – here you go.

Salting In:

In general, proteins tend to be more soluble when going from lower salt to medium salt (Figure 1). To give an estimated idea for these numbers, low salt is usually around 0 – 50mM and medium salt approximately 0.5M. The exact salt concentration will depend on the specific protein of interest and the particular salt being used, which we’ll discuss below in the Hofmeister Series section.

Figure 1. Salting In and Salting Out. As salt ions (pink and blue circles) are added to a solution, protein molecules (gray ovals) will first become more soluble (salting in). As more salt is added to the solution, the proteins will eventually start to aggregate (salting out).

Most proteins have charged patches on their surfaces. In the complete absence of salt, complementary charged patches will interact with one another, causing the protein to precipitate. As salt ions are added into the solution, those ions interact with the protein, preventing protein precipitation (Figure 2).

Figure 2. At low salt, complementary proteins (gray ovals) with charged surfaces (positive and negative signs) will bind to each other causing the protein to precipitate (left). At medium salt, the salt ions in solution (pink and blue circles) will interact with the charged protein surfaces and keep the protein soluble.

Salting Out:

As we keep adding salt to the solution, at some point we’ll pass the threshold of maximal solubility for our protein of interest (upper vertex in Figure 1). After that, adding salt will no longer help solubilize the protein, but instead will start causing the protein to precipitate.

In addition to charged patches, most proteins have hydrophobic patches on their surface. You can think of hydrophobic patches like grease – these patches don’t like to interact with water, or with salt ions. As the concentration of salt increases, these hydrophobic patches will clump together causing proteins to precipitate (Figure 3).

Figure 3. Proteins (gray triangles) with hydrophobic patches (orange ovals) will be more soluble at low salt (left). At higher salt, the hydrophobic patches from the proteins will bind together to avoid the salt ions (pink and blue circles) causing the protein to precipitate (right).

As we’ve described it so far, it might sound like all proteins will solubilize (salt in) and precipitate (salt out) at the same salt concentrations. However, this is definitely not the case. The schematic in Figure 1 is meant to give you a general idea of salting in and salting out, but the actual salt concentrations at which these processes occur depends on the nature of the specific protein that you’re interested in.

There is a lot research about what causes salting in and salting out at different salt concentrations for distinct proteins (He and Ewing, 2023; Okur et al, 2017). To simplify, it essentially comes down to the surface composition of distinct proteins. How many charged patches and hydrophobic patches does a given protein have? How large are those charged and hydrophobic patches, and how are they arranged on the protein’s surface?

As an extreme case – membrane proteins hate salt! They are extremely hydrophobic because normally most of the protein’s surface is surrounded by hydrophobic lipids. There is basically no salting in phase for membrane proteins, just a salting out phase (Figure 4) (Moringo et al, 2019).

By the way, a protein’s surface charge is not fixed, but is actually dependent on the pH of the solution that the protein is in. For the purpose of this article, we’re neglecting this fact and just assuming you’re maintaining a constant pH. But if you want to learn more about the relationship between pH and protein charge – check out this article.

Below, we’ll discuss how these differences in salting in and salting out behavior are used for protein purification. But first, we need to get into some specifics on the salt side of things.

Figure 4. For hydrophobic proteins, such as membrane proteins, there is really no salting in phase – they are most soluble at low or no salt. As the salt concentration increases, these proteins become less soluble as they clump together to hide their hydrophobic patches from the salt ions.

Hofmeister Series: Which Salt and How Much?

Just as the specific protein you’re working with matters, so does the type of salt that you are using to salt in or salt out your protein. The key takeaway from this section is that not all salts are created equal! Different salts have different potencies, so to speak. And that means the concentration of a salt needed for salting in or salting out a given protein will depend on the identity of the salt you are using.

This is an old idea that originates with Professor Franz Hofmeister’s studies in the late 19^th century. Hofmeister was using salts to precipitate proteins out of egg whites and noticed that different cations and anions had distinct potencies at precipitating egg white protein (Hofmeister, 1888). He rank-ordered the cations and anions into what are now known as the Hofmeister Series (Figure 5).

Figure 5. Hofmeister Series of ions. Cations (top, blue) and anions (bottom, pink) arranged according to their salting out (left) and salting in (right) potency as well as their protein stability and protein denaturation potency.

Let’s briefly go over some of these salts and their common uses in biochemical research.

Ammonium sulfate [(NH₄)₂SO₄] is frequently used for salting out proteins. Ammonium sulfate salts out many proteins, but at a variety of salt concentrations. Thus, using different concentrations of ammonium sulfate – informally referred to as ammonium sulfate cuts – are used to separate distinct proteins. See the next section for an example of how exactly this is done.

Sodium chloride (NaCl) and potassium chloride (KCl) are probably the most frequently used salts in biochemical research and are also commonly used to salt in proteins. However, there are a few proteins that behave poorly with chloride anions. Chloride is not present in high concentrations in most cells; there’s probably an extremophilic bacteria somewhere that loves chloride, but in most normal environmental conditions, chloride concentrations are low (Theillet et al, 2014). Acetate and the amino acid glutamate are more abundant cellular anions, and therefore more physiologically-relevant anions for proteins that don’t like chloride (Kozlov et al, 2017; Kozlov et al, 2022; Reichert and Moore, 2000).

Magnesium (Mg²⁺) and Calcium (Ca²⁺) are divalent metal cations meaning that they have a +2 charge and can interact with two different negative charges at the same time. These are important metal cofactors for many protein enzymes, and so they are frequently included in protein purification buffers at approximately 1 to 2mM concentrations. Their divalent nature, however, leads them to more potently denature proteins compared to sodium or potassium cations. This is because they can bind to negative charges on two different proteins at the same time thereby promoting intermolecular interactions (Figure 6).

Figure 6. Monovalent vs. divalent cations. Monovalent cations bind to negatively charged protein surfaces but can only bind to one negative charge at a time (left). Divalent cations can bind to two negative charges simultaneously and therefore can bridge interactions between two negative charges on different proteins leading to protein denaturation (right).

Lastly, guanidinium is very powerful at denaturing protein structure. This ion is used in cases when you want to denature every protein in a given sample. One example of this is refolding proteins from inclusion bodies.

Ok, now with a little more detail into some of these salts, I think you can see why not all salts are created equal. Picking a salt for your salting in or salting out experiment will determine how broadly the salt impacts different proteins, and the concentration regime of salt that you need to use for your desired effect.

Using Salts to Facilitate Protein Purification

As mentioned above, if every protein salted in and salted out at the same salt concentration, then this technique would be useless in terms of helping you purify your protein. However, many proteins will have different solubilities in response to distinct salt concentrations, and this solubility can be used to selectively purify your protein of interest.

Let’s consider a hypothetical example with six proteins:

• an orange circle and square
• a green rectangle and hexagon
• and a purple triangle and pentagon.

The proteins with the same color have similar salt solubility profiles. Relative to each other, the orange proteins are most soluble at low salt concentrations, the green proteins at medium salt concentrations, and the purple proteins at high salt concentrations (Figure 7).

Figure 7. Different proteins have different solubilities in relation to salt concentration. In this hypothetical example the orange proteins are most soluble at low salt, the green proteins at medium, and the purple proteins at high salt concentrations. The dotted gray lines indicate the salt concentrations for Figures 8, 9, and 10, as labelled.

As discussed above, based on these solubilities at different salt concentrations we would hypothesize that the orange proteins are enriched in hydrophobic surfaces, the purple proteins are enriched in electrostatic surfaces, and the green proteins are more of a mixture of each type of surface.

Their different solubilities can be used to purify away a subset of these proteins. Let’s say you wanted to purify the green hexagon. By increasing the salt to a medium-high level, you would precipitate the orange proteins, while the green and purple proteins would still be in solution. Now by taking this solution and pipetting it into a new tube, you have separated the green and purple proteins away from the orange proteins (Figure 8).

Figure 8. Orange proteins precipitate at medium-high salt concentration whereas green and purple proteins remain in solution and are separated by pipetting them to a new tube.

Now, to separate the green and purple proteins you have a couple of options. Personally, I would lower the salt to keep the green proteins in solution while precipitating the purple proteins (Figure 9). I would try this approach first because keeping your protein of interest (green hexagon) in solution is safer for making sure that you have functional protein in the end.

Figure 9. Purple proteins precipitate at low salt whereas the green proteins remain in solution and are separated by pipetting them into a new tube.

Alternatively, you could have raised the salt and precipitated the green proteins while keeping the purple proteins in solution (Figure 10). This works for separating the proteins – in this case you’ll just keep the pellet for the green hexagon. However, active and functional proteins cannot always be resolubilized from an insoluble pellet . So, depending on what exactly the protein of interest is, and what your intended downstream use for this protein, you may want to avoid precipitating it and take the low salt option in Figure 9 instead.

Whichever route you’ve taken, you have now separated the green proteins from the orange and purple proteins – nice! However, while you wanted the green hexagon, you still have the green rectangle hanging around too. That’s because salting in and salting out are partial purification techniques. They can eliminate some of the other proteins from a cellular extract – but you will probably need to use additional protein purification techniques to get really pure proteins.

So that’s all about salting in and salting out proteins! This can be a great initial purification technique, especially for native proteins that lack an affinity tag, and in combination with additional purification steps like ion-exchange and size-exclusion chromatography.