In all kinds of cell, protein, and DNA experiments, once the components leave the natural biological environment, they will be seriously affected. To provide them with a good natural environment, buffer must be used, TRIS, phosphate, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), etc...... then how to choose a most suitable buffer?
Let's take a look at the commonly used biological buffers, and their characteristics:
1. Phosphate buffer
Phosphate buffer is the most widely used buffer. Due to its secondary dissociation, the buffer has a wide pH range and can be configured with acidic, basic and neutral buffers of various pH values:
2. TRIS buffer
Tris buffer is widely used in biochemical research. It is a weak base and usually used in the "neutral" range. Tris-HCl buffer: pH=7.5~8.5; Tris-phosphate Buffer: pH = 5.0 to 9.0.
In addition to Tris-HCl, TRIS has a variety of derivatization buffers:
3. Glycine buffer
Glycine buffer has a wide pH range and a wide range of applications. The pH range is 2.0~11.0. The most common system and pH range are shown in the following table:
Glycine buffer |
pH range |
Glycine-HCl |
2.0~5.0 |
Glycine-NaOH |
8.0~11.0 |
Glycine-Tris |
8.0~11.0 |
Glycinamide buffer |
7.8~8.8 |
Glycylglycine buffer |
8.0~9.0 |
4. HEPES buffer
HEPES is a non-ionic amphoteric buffer with good buffering capacity in the range of pH 7.2~7.4. It is commonly used in biochemical diagnostic kits, DNA/RNA extraction kits and PCR diagnostic kits.
Table 1. Advantages and disadvantages of commonly used buffers
Buffer |
Advantages |
Disadvantages |
Phosphate |
Easy to prepare into various concentrations Wide pH range pH is less affected by temperature and concentration |
Easy to form precipitation with common calcium, magnesium and heavy metal ions inhibit certain biochemical processes |
TRIS |
Alkaline is strong; It has little interference with biochemical processes and does not precipitate with calcium, magnesium ions and heavy metal ions |
pH is greatly affected by concentration, diluted 10 times, pH change is greater than 0.1 Large temperature effect, such as pH=8.4 at 4°C and pH=7.4 at 37°C Easy to absorb carbon dioxide in the air Interference with some pH electrodes |
Glycine |
Provides a closer natural environment for cell components and various extracts |
Similar to the phosphate buffer system; interferes with certain biochemical reaction processes, such as metabolic processes; |
HEPES |
Maintains a constant pH value during open culture or cell observation and has no toxic effects on cells |
It may be toxic to some cells at high concentrations and slightly more expensive than other buffers. |
General principles for selecting buffers:
(1) The most important thing is to determine the pH value required by your system.
(2) Select the buffer system, the buffering capacity will decrease significantly when the pKa changes more than one pH unit;
(3) The buffer system should not affect the activity of the protein or the stability of the DNA, and avoid interference from other ions;
(4) Finally, we must also consider the effect of temperature: temperature not only affects the buffer, but also affects the cells. Most animal cells have a pH of 7.0 to 7.5 at 37 °C, and when the temperature falls to 0 °C, it can reach 8.0.
Edited by Suzhou Yacoo Science Co., Ltd.
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