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When would you use a Hepes buffer?

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When would you use a Hepes buffer?

The full name of HEPES is 4-hydroxyethylpiperazine ethanesulfonic acid, CAS is 7365-45-9, HEPES is often used in biological buffers, pH buffer range: 6.8-8.2, the main component of hepes buffer is hydroxyethylpiperazine ethyl Sulfuric acid, HEPES is an amphoteric buffer, which is soluble in water and does not form stable complexes with metal ions. It has a good buffering capacity in the pH range of 7.2-7.4. It is mostly used in biochemical diagnostic kits, DNA/RNA extraction kits and PCR diagnostic kits.

 

Used in a variety of biochemical reactions:

1. HEPES buffer is often used as a buffer reagent in the cell culture medium of various types of organisms, cell-cell adhesion, short-term cell aggregation and culture, and buffer for cleaning tissues and cells;


2. In protein research, PIPES is often used as the component and eluent of the binding buffer in cation exchange chromatography;


3. In DNA research, PIPES is used as a buffer for calcium phosphate and DNA precipitate formation system, and as a buffer for AFM and electroporation experiments.


4. In addition, HEPES has a certain interference to the reaction between DNA and restriction enzymes, and it is not suitable for Lowry's method to determine protein content.


5. HEPES buffer is often used in the research of organelles and highly variable, pH-sensitive proteins and enzymes, as well as biochemical diagnostic kits, DNA/RNA extraction kits and PCR diagnostic kits.


6. The reaction buffer, pre-hybridization buffer, and hybridization buffer for separating and analyzing RNA nuclear components; used for RNA and T4RNA. Molecular biology grade is used to label the 3'-end of RNA with T4 RNA ligase, separate and analyze the components of the reaction buffer, pre-hybridization buffer and hybridization buffer of nuclear RNA.

 

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HEPES related issues

1. The pH required for most cells is 7.2-7.4, but the appropriate pH for cell culture varies with the types of cells being cultured. Fibroblasts prefer a higher pH (7.4-7.7), while the passage of transformed cell lines requires acidity. pH (7.0-7.4). Since most culture fluids are buffered by the system of sodium bicarbonate (NaHCO3) and CO2, the CO2 concentration in the gas phase should be in equilibrium with the sodium bicarbonate concentration in the culture fluid. If the CO2 concentration in the gas phase or in the incubator air is set at 5%, the amount of NaHCO3 added in the culture solution is 1.97g/L; if the CO2 concentration is maintained at 10%, the amount of NaHCO3 added in the culture solution is 3.95g/L. The cell culture bottle cap should not be screwed too tightly to ensure gas exchange;

 

2. The pH value of the culture solution using HCO3 and CO2 buffer pair is unstable and tends to be alkaline after storage for a certain period of time. If the pH of the culture fluid changes too quickly, HEPES buffer can be added to the culture fluid to a final concentration of 10-25mM;

 

3. HEPES is an amphoteric buffer, mostly used in biological research such as oxidative phosphorylation, protein synthesis in a sterile environment, photosynthetic phosphorylation, CO2 fixation, etc.; HEPES has no effect on the substrates of metal ionase and is suitable for transmission In electron microscopy (TEM); in cell culture media, the advantage is that it can maintain a relatively constant pH value during open culture or cell observation. HEPES (25mM) can be used as a substitute for bicarbonate buffer, or as a Addition of bicarbonate buffer (10-15mM) to relieve the restriction of high-concentration CO2 culture environment. Dissolved CO2 and bicarbonate are also very important for good cell growth.

Pub Time : 2021-05-26 13:57:52 >> News list
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