With the development of protein chemistry and molecular biology, the development and application of protein drugs for the treatment of various diseases has become a hot spot in the development of the biomedical industry. Peptide and protein drugs have small side effects, strong activity, and have the effect of treating both the symptoms and the root causes. When we purify a 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. Therefore, several factors should be considered when designing buffers: pH, buffer system, salt ions, reducing agents, and stabilizers.

pH

Many experiments set the pH at 7.4 to mimic biological conditions, but if the protein of interest is not stable under these conditions, the pH needs to be changed to make it soluble in solution and not degraded. When the pH of the solution is near the isoelectric point pI of the protein, it will appear less soluble in the solution, because the protein surface has no net charge at this time, so it is easy to aggregate. Protein isoelectric point pI values ​​can be calculated quickly and easily using the ProtParam tool on the ExPASy website, simply by submitting the protein sequence.

buffer system

First of all, we need to ensure that the selected buffer system has buffer capacity at the set pH value, and its dissociation constant pKa value should be within one unit of the set pH value.

The second is to ensure that the buffer concentration is high enough to achieve the effect of the actual buffer solution. Generally, the concentration chosen is 20~100mM. It should be noted that the buffer system used cannot affect the activity of the protein. For example, phosphate will inhibit the activity of the kinase, so it should be thoroughly dialyzed before the reaction.

In addition, some buffer systems are very temperature sensitive, such as Tris-HCl buffer, if the buffer system is adjusted to pH 8 at 25°C, its pH will increase to 8.58 at 5°C and drop to 7.71 at 37°C , so, if the experiment is not performed at 25°C, it should be considered that this pH value may not be applicable in the experimental conditions.

salt ion

NaCl is included in many buffers to help maintain protein solubility and mimic physiological conditions, typically at a concentration of 150 mM, but different concentrations of salt ions may be required for different protein purification steps. Ion exchange chromatography is generally low-salt binding and high-salt elution. When binding, the salt concentration needs to be reduced to avoid the competition between salt ions and proteins to combine with the filler under high ionic strength, preventing the protein from flowing through the ion exchange column, so that the column It can bind the target protein; while hydrophobic chromatography is generally high-salt binding and low-salt elution.

reducing agent

If the target protein contains cysteine ​​residues, oxidation problems between residues may occur and may lead to protein aggregation. To prevent this, some reducing agents such as DTT, TCEP, and mercaptoethanol are often added to the buffer.

TCEP is the most stable of the three reducing agents, but also the most expensive. Usually, DTT is added to the buffer in the purification process, and TCEP is added to the buffer that stores the enzyme solution at the end. The reducing agent concentration is generally 5~10mM, which should be much higher than the protein concentration. DTT and mercaptoethanol are degraded at room temperature, so buffers with added reducing agents need to be stored at low temperature, or added with reducing agents at the time of use.

When using a reducing agent, make sure that the column material is compatible with it. For example, a high concentration of reducing agent will strip nickel from the nickel column and make the column darker and brown. Although the nickel column can be regenerated, the column capacity will be reduced. will be greatly affected.

stabilizer

Adding some stabilizers to the buffer can help improve protein solubility and stability during protein purification. Adding inert protein BSA to the buffer can stabilize the target protein to some extent, but it must be ensured that these added stabilizers do not interfere with the experiment; sometimes glycerol, polyethylene glycol, etc. are added to increase the buffer viscosity and help prevent protein Aggregation; in addition, the use of a small amount of surfactants and some ionic compounds such as sulfate, amino acid, citric acid, etc. can avoid ionic interactions between proteins and help proteins dissolve.

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