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The commonly used biological buffers and how should they be selected?

2019-07-02 来源:亚科官网
Biological buffer is a solution that keeps the pH of the solution relatively stable when a small amount of acid or alkali is added. Most cells can only be alive in a narrow pH range, and a buffer system is needed to resist the occurrence of pH changes during metabolism. In biochemical research work, buffer solutions are often used to maintain the pH of the experimental system.
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 different pH values:
  Preparation of acidic buffer can be directly used NaH2PO4 or KH2PO4, pH range of 1-5;
  Alkaline buffer can be directly used Na2HPO4 or K2HPO4, pH range of 9~12;
  The neutral buffer contains equal amounts of NaH2PO4 and Na2HPO4 or equivalent amounts of KH2PO4 and K2HPO4 solutions with a pH of 5.5-8.5.
Advantages: easy to prepare into various concentrations; wide pH range; pH is less affected by temperature;
Disadvantages: easy to form precipitates with common calcium, magnesium, heavy metal ions; inhibit some biochemical processes;
2. Tris buffer
Tris buffers are widely used in biochemical research. It is a weak base and is usually used in the "neutral" range. Tris-HCl buffer: pH = 7.5~8.5; Triphosphate buffer: pH = 5.0~9.0.
In addition to TRIS-HCl, TRIS has a variety of derivatization buffers:
 TBS=Tris-HCl+ NaCl+KCl, commonly used to clean immunostained tissue or Western blotting membrane in Western blotting;
  TBST=Tris-HCl+NaCl+tween20, a membrane buffer commonly used in Western Blotting;
 TE=Tris-HCl+EDTA, which has a protective effect on DNA bases and is commonly used for DNA stabilization and storage;
 TAE=Tris base + acetic acid + EDTA, is a buffer system widely used in short-segment DNA electrophoresis;
  TBE = three bases + boric acid + EDTA, suitable for long-term DNA electrophoresis, has a good separation effect on small fragments.
Advantages: Because of the strong basicity of Tris base, it is possible to use only one buffer system to prepare a buffer with a wide range of pH ranging from acidic to alkaline; has little interference with biochemical processes, and does not interact with calcium, magnesium ions and heavy metal ions precipitate.
Disadvantages: The pH value of the buffer is greatly affected by the solution concentration, the buffer is diluted ten times, and the pH value is greater than 0.1; the temperature effect is large, and the temperature change has a great influence on the pH value of the buffer, so it must be used. Formulated at temperature, Tris-HCl buffer prepared at room temperature cannot be used at 0 °C ~ 4 °C; easy to absorb CO2 in the air, so the prepared buffer should be tightly sealed. This buffer has some interference effect on some pH electrodes, so use an electrode compatible with Tris solution.
3. Good’s buffer
In the mid-1960s, in view of the fact that general buffer systems were not suitable for biochemical experiments, N.E. Good had to use artificial and synthetic methods to find specific buffer systems specifically for life science research. The buffers have following qualities:
1. pKa is between 6 and 8;
2. has high solubility in aqueous solution;
3. salt effect is small;
4. is not easily transported through cell membrane;
5. is not susceptible to temperature, ion composition, concentration and other factors affecting dissociation ;
6. Will not form a complex with metal ions, or the complex does not precipitate, does not interfere with biological activity;
7. Chemically stable.
The main advantage of Good’s buffer is that it does not interfere with and does not interfere with the biochemical reaction process, and has no inhibitory effect on enzyme chemical reactions, so they are specifically used for the research of organelles and highly variability, pH-sensitive proteins and enzymes.
The disadvantages are: 1 expensive, 2 pairs of biuret method and Lowry method for determining protein content, because they will darken the color of the blank tube.
4. Glycine buffer
Glycine buffer has a wide pH range and a wide range of applications. The pH range is from 2.0 to 11.0. The most common systems and pH ranges are shown in the table below:

Glycine buffer
pH range
Glycinamide buffer
Glycylglycine buffer

Advantages: Provide a more intimate natural environment for cellular components and various extracts.
Disadvantages: similar to phosphate buffer systems; interfere with certain biochemical processes, such as metabolic processes.
Related links: glycine