The influence of buffer pH and ionic strength on protein purification
Release time:
2021-10-26
Protein purification refers to the process of separating different proteins by ion exchange chromatography to purify the target protein. In addition to protecting the stability of the protein structure, the pH and ionic strength of the buffer also directly affect the adsorption of the ion exchanger to the protein. The protein purification process plays a key role.
The influence of buffer pH and ionic strength on protein purification
Buffer pH and ionic strength
Protein purification is a process of protein separation and purification. First of all, it is necessary to ensure the integrity of the protein structure and ensure the activity of the protein. It is necessary to maintain the pH value of the protein through a buffer and adjust the appropriate ionic strength (for example, the commonly used Tris buffer is added with NaCl to increase Salt concentration), too high salt concentration affects the dissolution of the protein and even causes the protein to precipitate. In addition, the pH value and ionic strength of the buffer also have an important influence on the adsorption of proteins and ion exchangers.
The effect of buffer pH on protein adsorption
There are two types of exchangers in ion exchange chromatography: cation exchangers and anion exchangers. The cation exchanger itself is bound with an anion group, which can replace cations and can absorb positively charged proteins to exchange with it, while anion exchangers are the opposite. When the pH of the buffer is equal to the isoelectric point of the protein, the static charge of the protein is 0 and it will not be adsorbed. The pH of the buffer is higher than the isoelectric point, and the free carboxyl group of the protein is negatively charged, which can be adsorbed by the anion exchanger; the pH of the buffer is low At the isoelectric point, the free amino group of the protein is positively charged and can be subjected to cation exchange chromatography.
Influence of buffer ionic strength on protein adsorption
Different proteins have different charges and different degrees of binding to the exchanger. In addition to changing the pH of the buffer, the ionic strength can also be changed to affect the adsorption of the protein and the exchanger, thereby separating different proteins one by one. Both colloidal ions and inorganic salt ions of protein can be adsorbed by the exchanger, which is competitive. For example, in Tris-NaCl buffer, if the ion concentration increases, the protein will not be easily adsorbed or will be easily eluted after adsorption. If the ionic strength is low, the protein will be easily adsorbed and not easily eluted.
In summary, the pH and ionic strength of the buffer can not only maintain the stability of the protein structure, but also adjust the charge of the protein micelles and the adsorption of the protein as an ion exchanger. Desheng is a manufacturer of biological buffers and can provide buffers such as Tris and MOPS for protein purification.
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