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Biobuffer is a solution that can keep the pH value of the solution relatively stable when adding a small amount of acid or alkali. Most cells can only operate in a very narrow pH range, and a buffer system is needed to resist the pH changes in the metabolic process. In biochemical research, buffer solution is often used to maintain the ph of the experimental system.
1. Phosphate buffer
Phosphate buffer is the most widely used buffer. Due to secondary dissociation, the buffer has a wide pH range and can be configured with acidic, alkaline and neutral buffers of different pH values:
Acidic buffer can be prepared by using NaH2PO4 or KH2PO4 directly, pH range is 1~5;
Alkaline buffer can be directly used Na2HPO4 or K2HPO4, pH range is 9~12;
The neutral buffer contains equal amounts of NaH2PO4 and Na2HPO4 or equal amounts of KH2PO4 and K2HPO4 solutions with pH values of 5.5~8.5.
Advantages: ① Easy to prepare various concentrations; (2) wide pH range; (3) pH is little affected by temperature;
Disadvantages: (1) Easy to precipitate with common calcium, magnesium and heavy metal ions; ② Inhibit some biochemical processes;
2. Tris buffer
Tris buffer is 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 derivative buffers:
TBS= TrIS-HCl + NaCl+KCl, which is commonly used to clean immunostained tissues or Western blotting films;
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, is often used for DNA stability and storage;
TAE=Tris base + acetic acid +EDTA is a buffer system widely used in short fragment DNA electrophoresis.
TBE= tri base +peng acid +EDTA, suitable for long-term DNA electrophoresis, has a good separation effect on small fragments.
Advantages: (1) Because of the strong alkalinity of Tris base, we can only use this buffer system to prepare buffers with a wide range of pH values from acidic to alkaline; ② It has little interference to biochemical process and does not precipitate with calcium, magnesium and heavy metal ions.
Disadvantages: (1) Buffer pH value is greatly affected by the solution concentration, buffer dilution ten times, the change of pH value is greater than 0.1; ② Temperature effect is large, temperature change has a great influence on the pH value of buffer, so it must be prepared at the operating temperature, trIS-HCl buffer prepared at room temperature can not be used in 0℃ ~ 4℃. (3) Easy to absorb CO2 in the air, so the preparation of buffer to cover tightly sealed. ④ The buffer solution can interfere with some pH electrodes, so the electrode compatible with Tris solution should be used.
In the mid-1960s, N.E. Wood found that conventional buffer systems were not suitable for biochemical experiments, and it was necessary to find human-designed and synthetic buffers for life science research. These buffers had the following characteristics:
1. PKa is between 6 and 8; 2. High solubility in aqueous solution; 3. The salt effect is small; 4. Not easily transported through the cell membrane; 5. The dissociation degree is not susceptible to temperature, ion composition, concentration and other factors; 6. It will not form complex with metal ions, or the complex will not precipitate, do not interfere with biological activity; 7. Stable chemical properties.
The main advantage of Good's buffers is that they do not participate in and interfere with biochemical processes, and do not inhibit enzymatic chemical reactions, etc., so they are specifically used in the study of organelles and highly volatile, ph-sensitive proteins and enzymes.
The disadvantages are: (1) it is expensive, and (2) it is not suitable for the biuret and Lowry methods for the determination of protein content, because they will make the color of blank tubes darker.