In biochemical experiments, buffer solution plays an indispensable role, which can resist the impact of a small amount of strong acid and alkali, maintain the pH value closest to the physiological environment. Buffer agent HEPES—a zwitterionic organic chemical buffering agent with an effective buffer range of 6.8~8.2, which can be used in a wide range of biochemical reactions. It is commonly used in the research of organelles and highly denatured pH-sensitive proteins and enzymes, as well as biochemical diagnostic kit, DNA/RNA extraction kits and PCR diagnostic kit.

Then, how to prepare and use HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)?

About the preparation of HEPES buffer solution, there are different views in separate documents. Depending on the application, one is HEPES+NaOH and the other is HEPES+salt. The specific methods are summarized as follows:

(1) 1M HEPES + NaOH, pH = 7.0 (500 mL)

119.15g HEPES is dissolved in 400mL distilled water, then add 0.5~1M NaOH aqueous solution to adjust the pH, then dilute to 500mL with distilled water, store at 4°C.

(2) HEPES + salt (500 mL)

HEPES 6.5g, NaCl 8.0g, Na2HPO4•7H2O 0.198g, adjust the pH value with 0.5M NaOH aqueous solution, dilute to 500mL.

There are two ways to use HEPES (CAS: 7365-45-9):

(1) According to the required concentration, it can be directly added to the culture medium, and then filter and sterilize: 2.38g HEPES was added to each 1000mL culture medium, adjust the pH to 7.2 with 1N NaOH, filter and sterilize. The concentration of HEPES is 10mmol/L.

(2) Prepare 100x stock solution (1mol/L), take 99mL culture medium and 1mL stock solution, the final application concentration is still 10mmol/L.

HEPES (Model No.: Q0005) belongs to Good's buffer and has the advantages of high solubility in water, membrane impermeability, chemical resistance and enzymolysis resistance, and it does not combine with metal ions. But it should be noted that the final concentration of HEPES is 10~50mmol/L. Under normal circumstances, 20mmol/L of HEPES could achieve the buffering effect, researchers should prepare the solution according to the actual situation with the commercial HEPES.