In many biochemical experiments, it is necessary to use Good’s buffer, which can resist the influence of a small amount of strong acid and alkali, and provide the most suitable pH value for the reaction system. The most commonly used buffers are Tris buffer and HEPES buffer. Many people do not know the difference between them. Desheng company will give you an introduction:

Good’s buffer series

Introduction and application

Tris buffer: Tris is weakly alkaline, pKa is 8.1 at 25℃, the effective buffer range of Tris buffer is between pH 7.0~9.2, and the commonly used pH values in biochemical experiments are 6.8, 7.4, 8.0 and 8.8. It often plays a buffer role in electrophoresis and gel experiments, and is widely used as a solvent for nucleic acids and proteins, and can also be used for the formation of intermediate fibers in nematodes. The derived buffers, such as TE, TAE and TBE, are often used for DNA stabilization, storage and extraction.

HEPES (4-hydroxyethyl piperazine ethanesulfonic acid), is a kind of amphoteric ion biological buffer, which belongs to Good's buffer, with an effective buffer range of 6.8~8.2, which can be widely used in a variety of biochemical reactions and used as a buffer reagent in some cell culture media. The final concentration is 10~50mmol/L, and the buffering capacity can be achieved when the medium contains 20mmol/L. In addition, HEPES buffer is often used in the research of organelles and highly denatured, pH sensitive proteins and enzymes, as well as biochemical diagnostic kits, DNA/RNA extraction kits and PCR diagnostic kits due to its non cytotoxicity.

Characteristics of two buffers

Tris buffer solution:

1. Tris buffer solution itself is alkaline. Only Tris HCl buffer system can be used to prepare buffer solution with pH range from acid to alkaline, and it can form a variety of buffer systems with other substances, such as TE, TBS, TAE, TBE, etc;

2. It has little interference on biochemical process and does not precipitate with Calcium, Magnesium and heavy metal ions;

3. It is greatly affected by the concentration of the solution, diluted ten times, and the change of pH value is greater than 0.1;

4. Generally speaking, the pH value decreases by 0.03 for each degree of temperature rise;

5. Easy to absorb CO₂ in the air.

HEPES buffer

1. pKa value is 6.0 ~ 8.0;

2. High solubility in water;

3. It has membrane impermeability and is not easy to penetrate the biofilm;

4. It has limited influence on biochemical reaction, can resist chemical action and enzymolysis, and does not form complex or precipitate with metal ions;

5. It has very low absorption of visible light and ultraviolet light

6. The influence of ion concentration, solution composition and temperature on dissociation is small;

Buffer preparation method

1. There are two ways to prepare Tris HCl buffer:

① Prepare 0.05mol/L Tris and 0.05mol/L HCL solutions respectively, and then mix them according to the volume listed in the common table. Because the diluted hydrochloric acid of standard concentration is not easy to prepare, the second method is often used;

② Take the preparation of 1L 0.1mol/L Tris HCL buffer solution as an example: weigh 12.11g Tris alkali to dissolve in 950mL-970mL deionized water, stir and drop 4N HCL, use pH meter to measure the pH value of the solution to the required pH value, and then add water to make up to 1L.

2. Preparation of HEPES buffer solution: one is pure HEPES + NaOH, the other is HEPES + salt according to the use:

HEPES + NaOH (500mL): 119.15g HEPES is dissolved in 400ml distilled water, 0.5-1m NaOH aqueous solution is added to adjust at least the required pH, and then the volume is fixed to 500ml with distilled water;

HEPES + salt (500mL): HEPES 6.5g, NaCl 8.0g, Na₂HPO₄·7H₂O 0.198g, adjust the pH value with 0.5M NaOH aqueous solution, and fix the volume to 500mL.

Tris and HEPES buffers produced by Desheng company have certain differences in buffer range, experimental temperature, characteristics and preparation methods. In specific experiments, the experimental conditions need to be carefully analyzed to select the most appropriate buffer system. In addition, it should be noted that in ion exchange chromatography, such as cation exchange column, positive Tris buffer should be avoided, while HEPES belongs to amphoteric ion buffer, and both anion and cation exchange chromatography are applicable.