The choice of buffer in the process of protein preparation and purification is very important, and only a suitable buffer can ensure the activity and structural stability of the protein.
When we purify the protein, the most important thing is to keep the protein from being inactivated during the purification process, or to minimize the loss of protein activity caused by the purification process. This means that the protein must remain soluble and active throughout the purification process.
Therefore, it is very important to design a protein purification buffer system that prevents protein degradation and polymerization, especially in the experimental results. The following factors should be considered when designing the buffer: pH, buffer system, salt ions, reducing agents and stabilizers. Each of these factors should be optimized according to your target protein, and the activity of the target protein should be used as the evaluation criterion for optimization.
In many experiments, the pH value is set at 7.4 to mimic biological conditions. If your protein is stable at this pH, that’s great! If not, you need to change the pH to find a state where the target protein is soluble in the solution and will not be degraded.
A rule of thumb is that the protein will not be easily soluble in a pH solution near its isoelectric point pI, because the protein has no net charge on the surface of the pH solution at its isoelectric point, and thus tends to aggregate. It is recommended to use the ProtParam tool to quickly and easily calculate the pI value of the protein isoelectric point, as long as the protein sequence is submitted.
Once the pH of the buffer is determined, you need to choose what kind of buffer system to use. First of all, when choosing a buffer system, make sure that the buffer system you choose does have a buffering capacity at the pH value you set. The selected buffer system should have a dissociation constant pKa value within one pH unit above and below the set pH.
Furthermore, make sure that the concentration of the buffer you use is high enough to achieve the effect of the actual buffer solution. Normally, the concentration selected is 20~100mM. What needs to be remembered is that the buffer system you use should not affect protein activity! For example, phosphate inhibits the activity of kinases, so it should be dialyzed off thoroughly before the reaction.
In addition, some buffer systems are very sensitive to temperature, such as Tris-HCl buffer. If you adjust the buffer system to pH 8 at 25°C, the pH will increase to 8.58 at 5°C and drop to 7.71 at 37°C. . Therefore, if you plan to store proteins at 4°C or perform experiments at 37°C, you should consider that the pH value adjusted at room temperature may not be applicable in the experimental conditions.
Many buffers contain NaCl to help maintain protein solubility and simulate physiological conditions. 150 mM NaCl is generally used.
In different protein purification steps, it may be necessary to change the concentration of salt ions. For example, if the protein is purified by ion exchange chromatography, it is necessary to reduce the concentration of salt ions (5~25mM) at the beginning to avoid high ionic strength and protein competition and binding of the filler, which can prevent the protein from flowing through the ion exchange column, thereby making The column can bind to the target protein, eluting and removing impurities.
Other types of chromatographic separation columns, such as gel filtration columns and Ni2+ affinity chromatography columns, may require higher salt concentrations. I generally use up to 500 mM NaCl for high-salt elution to prevent non-specific binding between the protein and the column. Finally, the target protein is replaced with a new buffer salt concentration system through the gel column.
If your protein contains cysteine residues, the oxidation between cysteine residues may become a trouble and may lead to protein aggregation. To prevent this, some reducing agents such as DTT, TCEP or mercaptoethanol are often added to the buffer.
In general, TCEP is the most stable of these three reducing agents, but it is also the most expensive. I usually add DTT to the buffer in the purification process, and add TCEP to the buffer that holds the enzyme solution at the end. Generally a reasonable concentration of reducing agent is 5~10mM. Basically you have to make sure that the concentration of reducing agent is much higher than your protein concentration. Because DTT and mercaptoethanol are degraded at room temperature, it is necessary to store the buffer solution added with reducing agent at low temperature, or add reducing agent before use.
When using, make sure that the column material is compatible with these reducing agents. For example, a high concentration of reducing agent will remove the nickel from the nickel column and make the column darker and brown. At this time, you can choose a high-resistant nickel column. Although the column is easy to regenerate, your protein loading capacity Will be greatly affected. Many column materials will list the maximum concentration of reducing agent they can withstand, but I think it will affect more or less, not whether it reaches the maximum concentration value.
Finally, some stabilizers can be added to the protein purification buffer to help improve the solubility and stability of the protein purification. Adding inert protein BSA to the buffer can stabilize the protein to some extent, but it must be ensured that the added stable protein does not interfere with the experiment. Sometimes additives such as glycerol and polyethylene glycol can increase the viscosity of the buffer and help prevent protein aggregation. In addition, using a small amount of surfactants and some ionic compounds such as sulfates, amino acids, citric acid, etc. can shield the ionic interactions between proteins and help them dissolve.
By adjusting the pH, buffer system, salt ion, reducing agent and stabilizer of the protein purification buffer, a complete protein purification buffer system can be established, which can maintain the activity and stability of the protein during the purification process! This production adjustment is very conducive to the development of the production process for the separation and purification of protein products, enzyme preparation products and antibody drug products!
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