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Polyacrylamide gel electrophoresis is an electrophoresis method using a polyacrylamide gel as a supporting medium. On this support medium, it can be separated according to the molecular size of the separated substance and the molecular charge.

Polyacrylamide gels have the following advantages:

1 Polyacrylamide gel is a macromolecule composed of acrylamide and N, N' methylidene bis acrylamide. The gel has a lattice of a carbon-carbon polymer with an amide side chain and no or little pendant side groups, so the electroosmotic effect is relatively small and it is difficult to interact with the sample.
    2 Because polyacrylamide gel is a synthetic substance, the concentration ratio of monomer can be adjusted before polymerization to form different degrees of interchain structure, and the porosity can be varied within a wide range, which can be separated according to The size of the material molecule, choose the appropriate gel composition, so that it has both suitable void content and better mechanical properties. In general, a gel containing 7-7.5% of acrylamide is suitable for separating molecular weights ranging from 10,000 to 1 million substances, and proteins below 10,000 are using gels containing 15-30% of acrylamide. Especially large, 4% acrylamide-containing gel can be used. The macroporous gel is brittle, and the small pore gel is difficult to remove from the tube. Therefore, when the concentration of acrylamide is increased, the double acrylamide can be reduced to improve the gel. Mechanical behavior.
    3 Polyacrylamide is thermally stable in a certain concentration range. The gel is colorless and transparent, easy to observe, and can be directly measured by a detector.
    4 acrylamide is a relatively pure compound that can be refined to reduce pollution. The starting materials for the synthetic polyacrylamide gel are acrylamide and methylene bisacrylamide. Acrylamide is referred to as a monomer, and methylene bisacrylamide is referred to as a crosslinking agent. In an aqueous solution, a monomer and a crosslinking agent form a gel by a radical-initiated polymerization reaction.

In the reaction process of polyacrylamide gel formation, a catalyst is required, and the catalyst includes two parts of an initiator and a further accelerator. The initiator provides the initial radical in the gel formation, and the acrylamide becomes a radical by the transfer of the radical, and the polymerization reaction is started, and the accelerator accelerates the rate of the radical release of the radical. The compatibility of commonly used initiators and accelerators is as follows:

Polymerization catalyst compatibility

　　注：(NH₄)₂S₂O₈，过硫酸胺 TEMED：N，N，N，N';四甲基乙二胺 DMAPN：β－二甲基胺基丙晴 　　The reaction initiated by ammonium persulfate is called chemical polymerization; when it is initiated by riboflavin, it is required to irradiate the reaction solution with strong light, which is called photopolymerization. The polymerization of polyacrylamide can be affected by the following factors: 1. The oxygen in the atmosphere can quench the free radicals and terminate the polymerization reaction, so the reaction liquid should be isolated from the air during the polymerization. 2. Certain materials, such as plexiglass, can inhibit polymerization. 3. Some chemical drugs can slow down the reaction rate, such as red blood salt. 4. The temperature is high and the polymerization is fast, and the temperature is low and the polymerization is slow. The above points must be taken care of when preparing the gel. The mesh size, mechanical strength and transparency of the gel are largely determined by the concentration and crosslinking of the gel. The total grams of monomer and crosslinker per 100 liters of gel solution is called gel concentration, which is usually expressed by T%; the percentage of crosslinker in the gel solution as a percentage of the total amount of monomer and crosslinked body is called Union degree, commonly used C%, can be calculated by:

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a: gram of acrylamide; b: grams of bisacrylamide; m: buffer volume (ml) When the gel concentration is too high, the gel is hard and brittle and easily broken; when the gel concentration is too low, the gel It is soft and difficult to operate.

The degree of cross-linking is too high, the glue is opaque and lacks elasticity; the degree of cross-linking is too low, and the gel is mushy. The polyacrylamide gel has a high viscosity, it does not prevent convection from reducing the ability to diffuse, and because it has a three-dimensional network structure, the ability of a molecule to pass through such a mesh will depend on the gel pores and the particles of the separated material. The size and shape of this gel is the molecular sieve effect of the gel. Due to the molecular sieve action, the gel here is not only a simple support. Therefore, in addition to paying attention to the basic principles of electrophoresis in the electrophoresis process, attention must also be paid to various properties related to the gel itself (the size of the mesh and Shape, etc.). The appropriate gel mesh can be selected by the following formula.

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Where: P is the average diameter of the mesh, C is the concentration of the polymer, d is the molecular diameter of the polymer (if the molecule is not crimped, it should be 5A), K is a constant, and the value of K depends on the geometry of the rubber. If the chain of the polymer is crosslinked at approximately a right angle, then about 1.5, according to this formula, we can approximate the mesh diameter by the polymer concentration C, for example, the known polymer concentration is 5%. The average diameter of the mesh should be:

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This calculation is rough and has a certain distance from the actual situation. Some people have determined the total concentration (T) of acrylamide solution, and the size of the mesh after polymerization in the presence of six different ratios of bisacrylamide. The pore size is related to the total concentration. The larger the total concentration is, the smaller the pore diameter is, and the mechanical strength is enhanced. When the total concentration is constant, the concentration of the bis-bisacrylamide (Bis) is the smallest at 5%, higher or lower. At the time of the value, the pore size of the polymer is relatively large, and the gel pore size is an important parameter in gel electrophoresis, which often determines the separation effect of electrophoresis. After continuous practice, the empirical values ​​as shown in Table 3 were obtained. In general, the protein in most organisms uses a 7.5% concentration gel, and the resulting electrophoresis results are often satisfactory, so it is called the concentration. The gel is a "standard gel."

For those gels used in important studies, it is best to pre-test by using a 10% series of gel concentration ladders to select the optimum gel concentration.

Table 3 Percentage of gel concentration selected for gel electrophoresis of proteins and nucleic acids of different molecular weight ranges

　　Polyacrylamide gel electrophoresis can be divided into two types: continuous and discontinuous. The former refers to the buffer used in the whole electrophoresis system. The pH value and the gel mesh are the same. The latter refers to the use in the electrophoresis system. Two or more buffers, pH and pore size, and discontinuous electrophoresis enable the dilute sample to be concentrated into layers during electrophoresis to improve resolution.

When a protein is electrophoresed in a polyacrylamide gel, its mobility depends on the net charge it carries and the size and shape of the molecule. If a reagent is added to eliminate the charge factor, the electrophoretic mobility depends on the size of the molecule, and the molecular weight of the protein can be determined by electrophoresis. In 1967, Shapiro et al. found that the anionic detergent sodium dodecyl sulfate (SDS) had this effect. When a sufficient amount of SDS and mercaptoethanol are added to the protein solution, SDS can reduce disulfide bonds in the protein molecule. Because the dodecyl sulfate is negatively charged, the various protein-SDS complexes are carried with the same density of negative charges, and its amount greatly exceeds the original molecular charge of the protein molecules, thus masking the original between different kinds of proteins. The difference in charge, SDS combined with protein, can also cause conformational changes. The protein-SDS complex forms a long-elliptical rod with a shape similar to "cigar". The short-axis length of SDS complexes of different proteins is different, about 18A. The mobility of such a protein-SDS complex in a gel is no longer affected by the charge and shape of the protein, but depends on the molecular weight, so SDS polyacrylamide gel electrophoresis can be used to determine the molecular weight of the protein. .

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