How do you make a sodium HEPES buffer?

HEPES Stock Solution (0.1 M, pH 7.4) Add 2.38 g of HEPES to an appropriate beaker (100-200 mL beaker in this case). Add about 80 mL of deionized water to the beaker. Add a stir bar to the beaker and leave it on a stir plate until completely dissolved (~1 min). Begin monitoring pH of the solution.

How do you make a HEPES buffer?


  1. Add 119.15 g HEPES (free acid) to a suitable container and make up to 400ml with distilled water.
  2. Add solid NaOH a few pellets at a time while mixing until the pH is ~6.8.
  3. Add concentrated NaOH dropwise to achieve pH = 7.0.
  4. Add distilled water to a final volume of 500 ml.
  5. Sterile filter and store for later use.

Does HEPES buffer expire?

HEPES sodium salt is stable at least three years if stored sealed and kept dry at room temperature. Although Sigma does not assign expiration dates, sodium HEPES should be re-evaluated for continued suitability in user application every three to five years.

How does a HEPES buffer work?

HEPES is widely used in cell culture, largely because it is better at maintaining physiological pH despite changes in carbon dioxide concentration (produced by aerobic respiration) when compared to bicarbonate buffers, which are also commonly used in cell culture.

What pH is HEPES?

6.8 to 8.2
HEPES has been described as one of the best all-purpose buffers available for biological research. At biological pH, the molecule is zwitterionic, and is effective as a buffer at pH 6.8 to 8.2 (pKa 7.55). It is typically used in cell culture at concentration between 5mM to 30 mM.

Is HEPES buffer stable?

Notes: If precipitation is present in the concentrated buffer, gently warm the bottle to 37 °C and mix until completely dissolved prior to dilution. Stable for two years from the date of manufacture when stored at room temperature.

Does HEPES buffer degrade?

Tips for HEPES handling and storage About the sterilization method of HEPES, HEPES powder is resistant to high temperatures with its melting point reaching 200℃, thus, it will not get degraded by autoclaving, or you can filter HEPES Buffer solutions by a 0.2-micrometer syringe filter.

When would you use a HEPES buffer?

Compared with other buffers such as PBS (phosphate buffered saline) and TRIS, HEPES has higher stability in maintaining the pH values of the cell culture media, that’s also the reason why HEPES is widely used in cell culture , tissue culture, protein purification and extraction, immunoprecipitation, cell lysis, live …

What is a good buffer range?

Buffers should have a pKa between 6.0 and 8.0 because the optimal pH for most biological reactions rests in this range. Buffers should have high water solubility and minimum solubility in organic solvents so it remains in the aqueous medium of the biological system. Buffers should not permeate cell membranes.

How to make a Hepes buffer with NaOH?

Procedure 1 Dissolve HEPES in about 800 mL of deionized water. 2 Adjust pH to 7.5 with NaOH. 3 Add deionized water to 1L.

How to prepare a 1 m, 7.5 pH Hepes buffer?

Prepare 800 mL of dH2O in a suitable container. Add 238.3 g of HEPES to the solution. Adjust solution to desired pH by 10N NaOH. Add dH2O until volume is 1 L. “HEPES Buffer (1 M, 7.5 pH) Preparation.”

How do you test Hepes buffer for transfection?

Test the formulation before use with transfection experiments. Test by mixing 0.5 ml of the above buffer with 0.5 ml 250 mM CaCl2 and vortexing. A fine precipitate should develop that is readily visible in the microscope. If this precipitate does not form, do not use the batch of buffer for transfection experiments.

Can you use HEPES as a protein deposition buffer?

HEPES is not recommended for certain protein applications such as the Folin-Ciocalteu protein assay; however, it does not affect the Biuret protein assay. HEPES is the buffer of choice in a protein deposition technique in electron microscopy because it did not affect metal substrates.

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