What attention should be paid to when using Tris-HCL buffer for protein purification?

Recently, many laboratories will use biological buffers in the protein purification process, and the selection of suitable buffers is particularly important at this time. Many people will choose Tris especially, thinking that the ionic strength is low, the buffering capacity is strong, its pH value is close to the physiological range, and it is a good solvent for proteins. But in fact, Tris-HCL buffer can also be used for protein purification, but in order to ensure the purification effect, some problems still need to be paid attention to during the experiment.

I. Pay attention to the concentration of Tris-HCL

In the purification experiment, the pH value of the buffer and the buffer system of Tris-HCl are determined, and the pH buffering effect of its set can be known. Usually, the dissociation constant pKa of the buffer has a buffer within one unit of the set pH value. effect. Only the combination of buffer range and dissociation constant is far from enough. In addition, it is necessary to ensure that the concentration of buffer Tris-HCl is high enough to achieve practical effect. The general concentration is 20-100mM.

II. Pay attention to the sensitivity of Tris-HCL to temperature

Tris series buffer systems are sensitive to temperature. For example, when Tris-HCL is adjusted to pH 8 at 25°C, the pH value will increase to 8.58 at 5°C, and drop to 7.71 at 37°C. Therefore, when storing proteins at 4°C or performing experiments at 37°C, consideration should be given to whether the pH lowered at room temperature may be applicable in the experimental conditions.

III. Pay attention to the salt in the Tris-HCL buffer

Tris-HCL buffer is used in protein purification experiments, and sodium chloride needs to be added to increase the salt concentration to help maintain protein solubility and simulate physiological conditions. The commonly used concentration is 150mM. At the beginning of protein purification by ion exchange chromatography, it is necessary to reduce the salt concentration to reduce competition, and the concentration of gel filtration column and Ni2+ affinity chromatography column needs to be increased. The buffer system will not interfere with the activity of the protein. Tris-HCL cannot contain phosphate, otherwise the activity of some proteins will be inhibited, and it will be completely dialyzed before the reaction.

Since its establishment, Desheng Biochemical has many years of experience in the production of biological buffers, and can provide large quantities of buffers such as Tris, Bicine, and MOPS. And for product quality, the company has specially set up a quality department to repeatedly sample and test batches of products before they leave the warehouse. If you have a demand for biological buffers, you can purchase with confidence, Desheng will escort you, please click on the website for consultation!