All About the Composition of Protein Purification Buffers and Why It Matters

Protein purification buffers are complex and can contain reducing agents, detergents, salts, imidazole, chelators, protease inhibitors, osmolytes, co-factors, and stabilizers. These chemicals maximize protein yield while maintaining integrity, and sometimes functionality as well.

Buffers for protein purification are designed very carefully to achieve three important objectives in protein experiments:

• They ensure that the protein purification procedure leads to high yields of the target protein, and the protein is not degraded during the purification process. In some cases, where downstream experiments require that the purified protein retains its function, the buffer is chosen to prevent protein denaturation while being purified.
• The purified target protein is free of contaminants, such as other proteins, nucleic acids, lipids and salts.
• The buffer used in purification, is compatible with downstream experiments. For example, imidazole in buffers can be a problem if the downstream step requires assaying by Lowry method. In this case, you would either have to use a different buffer or have a different protein estimation method.

To achieve these goals, a wide range of carefully chosen chemicals are used to prepare protein purification buffers.

In the first section of this article, we will discuss common types of chemicals used in these buffers, along with their intended purposes and considerations.

Then, to put things more into perspective, we will take a closer look at a specific purification buffer – the His-tag buffer, to see an actual example of a purification buffer and how its constituents help with protein purification.

Finally, we will describe a few common buffers used in protein purification and some considerations about using them in your experiment.

Article Table of Contents

Understanding the Composition of Protein Purification Buffers

Constituents of protein purification buffers

Chemicals for pH maintenance and why they matter

Salts for ionic balance/ stability

Detergents in protein purification buffers

Reducing agents in protein purification buffers

Protease inhibitors in protein purification buffers

Protein stabilizers in protein purification buffers

Chelating agents in protein purification buffers

His-tag buffer set

References

Constituents of protein purification buffers

Chemical components of protein buffers include reagents like Tris or HEPES, salts, detergents, reducing agents, protease inhibitors, imidazole and protein stabilizers. These constituents help maintain pH, denature or solubilize proteins, minimize damage and help maximize yield.

The chemicals that are used in common protein purification buffers, based on their utility in the purification procedure, may be classified into one or multiple of these categories:

• Chemicals for pH maintenance
• Salts for ionic balance and stability
• Detergents for denaturing and solubilizing the target protein
• Reducing agents for preventing oxidative damage to the target protein
• Protease inhibitors to inhibit protein degradation
• Co-factors to aid in stabilizing the target protein
• Osmolytes and other stabilizing chemicals that may help with both solubilizing and stabilizing the target protein
• Chelating agents that serve a wide range of purposes

Let’s take a closer look at each of these categories.

Chemicals for pH maintenance and why they matter

All protein purification buffers are designed to maintain a narrow pH range – with the help of chemicals like Tris, Tris-HCl and salts. This is important so that the target is stable, functional and that your protein is purified properly – especially in affinity or ion-exchange purification.

A primary function of any buffer is to maintain the pH of the experimental environment to a very narrow range.

pH maintenance in the experiment is critical during protein purification procedures for several reasons:

Ensure target proteins bind to the column:

You need to maintain an optimal pH to ensure that the target protein can bind to the purification column – during affinity purification.

Proteins ionize at a certain pH:

If you are using ion-exchange purification, you would need to maintain a specific pH at which the target protein ionizes in a way that is compatible with the ion-exchange column you are using.

For example, if your column is negatively charged, you would want the target protein to be positively charged during the purification process so that it binds to the negatively charged column.

Conversely, if you are using a positively charged column, you want your target protein to be negatively charged. And, how a specific protein ionizes, is dependent on the pH of the experimental conditions – which the buffer determines. Here is an article where you would get a whole lot of information about pH, how buffers regulate the pH of the experimental conditions and why pH is so very important for protein experiments.

Coming back to ion-exchange purification, you start with a negatively charged column. And the target protein is suspended in a buffer (buffer1 in this case) that maintains a pH at which this protein is positively charged. You have another buffer (buffer 2), that maintains a pH at which the target protein would be negatively charged.

The cell lysate is prepared in buffer 1, and when applied to the column, the positively charged target proteins bind to the negatively charged column, and the non-specific unwanted proteins do not bind to the column and are washed away.

After this, the column is treated with buffer 2 in the elution step. In buffer 2, the target protein becomes negatively charged and because of this – they are repelled from the negatively charged column.

Figure 1. The rectangle shows a negatively charged column. During the first stage, buffer 1 is applied and maintains the target protein (green) at a positive charge, attracting the protein to the column. After washing away nontarget proteins, buffer 2 is applied and maintains a negative charge for the target protein, repelling the target away from the column – this way, the target protein is eluted from the column.

Maintains compatible pH with downstream experiments:

It is important that the pH of the medium in which you finally purify your target protein is compatible with downstream experiments. For example, you will want to purify your protein in acidic conditions if the downstream experiments require an acidic pH.

Long story short – you should choose a buffer whose pK is +/- 1 of the pH you need for your purification.

Salts for ionic balance/ stability

Salts are universal components of protein purification buffers. Common examples of salt ions for protein purification buffers are Na⁺, K⁺, NH4⁺, Ca²⁺, Mg²⁺, Cl^-, PO₄^3-, SO₄^2-, HPO₄^2-, H₂PO₄^-. They provide the required ionic concentration to optimize protein solubility. Also, some salts help with pH maintenance.

Salts are known to impact protein solubility a lot. At low ion concentrations, in the range of 0.5M or less, in general, protein solubility increases with increasing ion concentration. This is especially true for cytoplasmic proteins.

There might nevertheless be exceptions. For example, salting in, might be trickier for insoluble proteins like those in the membranes – these proteins might be less stable as you increase the salt concentration. And you would need to fine tune the purification buffer and the purification protocol based on the specific needs of your experiment.

This is technically known as the salting in of proteins – where the salt ion concentration of the buffer is increased to solubilize the target protein.

Figure 2. Schematic representation of salting-in and salting-out of proteins. At low salt ion concentrations, solubility of the target protein increases with increasing concentration of the salt. At high salt ion concentration, solubility of the target protein decreases with increasing salt concentration.

Later in the purification procedure, when you want to recover the target protein from the solution, you can precipitate it by a technique known as salting out – built on the phenomenon that protein solubility decreases with increasing salt concentration, when the ionic strength is high.

While salting in and salting out might be relatively straightforward with cytoplasmic proteins, these steps might be far more nuanced when dealing with more complex cases – insoluble membrane proteins for example.

In any situation, how exactly you’d want to design the experimental steps for salting in and out depends on the specific characteristics of your target protein and the needs of your experiment overall.

Salts in the buffer are also important in the wash and elution steps of protein purification using both affinity and ion-exchange columns.

Though not a salt, chemicals like imidazole are used in protein purification buffers for a similar purpose. We will describe the role of Imidazole later in this article.

Detergents in protein purification buffers

Detergents are used in buffers when purifying poorly soluble or insoluble proteins like membrane proteins.

Detergents like SDS also aid in denaturing proteins to their linear structures – used if the tertiary functional structure of the target protein is not required in the purification.

Detergents in protein purification buffers, depending on what you need in the experiment, may be denaturing (like SDS) or non-denaturing, like CHAPS.

You will want to use non-denaturing detergents when you want to purify the target protein in its three-dimensional functional form. Detergents may also be categorized as cationic, anionic or neutral.

Reducing agents in protein purification buffers

Reducing agents like BME, DTT, TCEP and others are used in protein purification buffers to prevent oxidative damage to the target protein during the purification process.

Reducing agents also break disulfide bridges in proteins. So, they help in partially denaturing proteins during purification.

Figure 3. Reducing agents such as DTT/ BME break disulfide bridges between cysteine residues in a protein. Since disulfide bridges are a type of tertiary structure in proteins, this helps with protein denaturation.

You need to careful about whether you want reducing agents in the protein purification buffer for two reasons. One, whether you want to purify the target protein in a functional or denatured condition – reducing agents denature proteins. Two, whether the downstream steps following purification are compatible with the reducing agents you have in the buffer.

Finally, if you are absolutely sure that the protein you are purifying does not have any disulfide bridges, you might consider not adding any reducing agent because reducing agents help with denaturation only when the protein has a disulfide bond in its tertiary structure.

Protease inhibitors in protein purification buffers

It is common to have protease inhibitors in buffers for protein purification. During purification, proteins are very prone to degradation. To prevent this, protease inhibitors like PMSF and protease inhibitor cocktails are added to the buffer.

When a protein is being purified, it can get in contact with proteases when the cells are lysed. There also might be proteases in the experimental media or surfaces due to accidental contamination. Protease inhibitors prevent the target protein from protease degradation.

Figure 4. Protease inhibitors are commonly added to protein purification buffers to prevent unwanted degradation of the target protein. The utility of protease inhibitors is illustrated in this figure.

Though, not as commonly used as protease inhibitors, phosphatase inhibitors are also added sometimes in the buffer during purification – especially, when you want to purify the target protein in its native phosphorylation state.

Protein stabilizers in protein purification buffers

Purification processes are often harsh for proteins, and involve chemicals like sugars, amino acids, TMAO (Trimethylamine n-oxide), Trifluoroethanol (TFE), and glycerol for stabilization. Co-factors such as NAD⁺ and GTP are also added to the purification buffer to enhance protein stability.

Compounds that promote protein stability and folding during periods of stress are called osmolytes.

Cells, during stressful or harsh conditions, accumulate osmolytes to maintain cellular protein homeostasis. Some osmolytes are used in buffers during protein purification to increase stability of the target protein.

Here are some examples. TMAO is used as a stabilizer, especially when the purification process involves urea, which has destabilizing effects on proteins (Liao et al. 2017).

TFE is used, when the alpha helices in the target protein need to be stabilized. Glycerol, amino acids like glycine, sugars and other polysaccharides may be used as stabilizing chemicals, depending on the specifics of your experiment.

Co-factors such as NAD⁺, ATP, GTP and metal ions like Mg²⁺ impact protein structure and stability. These chemicals are added in the purification buffer especially when you want to purify a protein in its properly folded functional state.

Chelating agents in protein purification buffers

Chelating agents bind to divalent cations like Ca²⁺ and Mg²⁺ and remove them from the solution. Reducing metal ion concentration by chelation is done to inhibit proteases and to regulate target protein solubility.

They are common additives in protein purification buffers. EDTA is the most commonly used.

Deciding to use a chelating agent in protein purification depends on various factors, like whether the chelator works well with the target protein and the chosen purification method (such as an affinity column). It also depends on whether other steps in the process allow the use of chelating agents.

Now that we’ve seen the components and additives that are generally present in a protein purification buffer, here is a specific example of a buffer used in protein purification that will help you put into perspective what we discussed.

His-tag buffer set

The His-tag buffer set is optimized for using while purifying histidine-tagged proteins with a Ni²⁺ or Co²⁺ affinity column. It contains chemicals for maintaining pH and ionic balance, as well as for ensuring that the target protein binds to the column and is later eluted out.

In line with what we saw in the last section, the buffers in this set maintain the pH around 8. They also contain 300mM NaCl – ensuring correct ionic balance of cations (Na⁺) and anions (Cl^-) in the purification process.

In addition to this, one of the two buffers in the set – the one used for equilibrating the column and washing, has a low concentration (10mM) of Imidazole.

This is helpful because imidazole competes with the his-tagged target proteins to bind to the metal affinity column. The wash buffer contains a very low concentration of Imidazole (10mM). While this concentration of Imidazole is sufficient to prevent non-specific protein binding (that are not his-tagged) to the column, the target proteins (which are his-tagged) can easily outcompete with imidazole in binding to the column.

Figure 5. Represents a look inside the column where a low concentration of imidazole (blue) prevents nonspecific binding of nontarget proteins (pink/orange). The concentration is low enough for the His-tagged protein (green) to bind to the nickel bead.

The second buffer in the set, used in eluting out the target protein from the column, has a much higher concentration (500mM) of imidazole. The elution buffer has a large concentration of imidazole (500mM), which then dislodges the his-tagged proteins bound to the column, and you get your purified target protein eluting out from the column.

Figure 6. Shows the inside of a column where this buffer contains a higher imidazole (blue) concentration. Imidazole, at this concentration, outcompetes his-tagged proteins, dislodging them from Nickel beads and enabling elution.

As we saw that components in your buffer can interfere with downstream experiments, imidazole in this buffer makes it incompatible with the Lowry method of protein estimation. That being said, this buffer is compatible with most detergents and other additives.

To sum it all up, here are the key points you need to be aware of when choosing or designing a buffer for your protein purification experiment – the pH you need, the ions and their concentrations, whether you want to use detergents or reducing agents, the type of protease inhibitors you want, and finally, if your target protein requires stabilizers.

For every protein purification experiment – it is about maximizing your target protein yield with as minimal contamination and degradation as possible. And the buffer you choose helps in a large way to achieve your objective.

References

• Duong-Ly and Gabelli. 2014. Salting out of proteins using ammonium sulfate precipitation. Methods Enzymol. 2014:541:85-94
• Liao et al. 2017. Trimethylamine N-oxide stabilizes proteins via a distinct mechanism compared with betaine and glycine. PNAS. 114 (10) 2479-2484
• Shiraki et al. 1995. Trifluoroethanol-induced stabilization of the alpha-helical structure of beta-lactoglobulin: implication for non-hierarchical protein folding. J Mol Biol. 13;245(2):180-94
• Hauptmann et al. 2018. Impact of Buffer, Protein Concentration and Sucrose Addition on the Aggregation and Particle Formation during Freezing and Thawing. Pharm Res. 35(5): 101
• Vagenende et al. 2009. Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry. 48(46):11084-96