1807 was first discovered by Ferdinand Frederic Reius of Moscow University in Russia.
During the period of 1936, Swedish scholar A.W.K Tiselius designed and manufactured a mobile interface electrophoresis instrument, separated three kinds of globulin of horse serum albumin, and established electrophoresis technology.
Chinese name: electrophoresis mbth: electrophoresis proposed by Ferdinand Frederick Royce Author: 1807 Applied disciplines: chemical electrophoresis phenomenon, charge movement law, application field, electrophoretic paint film, electrophoresis type, moving interface electrophoresis, zone electrophoresis, isoelectric focusing electrophoresis, isokinetic electrophoresis, electrophoresis principle, review, electrolysis, electrophoresis, electrodeposition, electroosmosis, etc. Under certain conditions, the distance (i.e. mobility) that charged particles move in unit time under the action of unit electric field intensity is constant, which is the physical and chemical characteristic constant of charged particles. Different charged particles electrophorese in the same electric field because of different charges or different charge-mass ratios, and after a certain period of time, they separate from each other because of different moving distances. The separation distance is proportional to the applied electric field voltage and electrophoresis time. Under the action of external DC power supply, colloidal particles move directionally to cathode or anode in dispersion medium, which is called electrophoresis. Separation of substances by electrophoresis is also called electrophoresis. The colloid has electrophoresis phenomenon, which proves that the particles of colloid are charged. Various colloidal particles have different properties and absorb different ions, so they have different charges. The electrical properties of colloidal particles can be determined by electrophoresis. Colloidal particles moving to the anode are negatively charged, while colloidal particles moving to the cathode are positively charged. Generally speaking, colloidal particles such as metal hydroxide and metal oxide adsorb cations and are positively charged; Colloidal particles such as nonmetallic oxides and nonmetallic sulfides adsorb anions and are negatively charged. Therefore, in the electrophoresis experiment, iron hydroxide colloidal particles move to the cathode, and arsenic trioxide colloidal particles move to the anode. Sols with different charges can be separated by electrophoresis. For example, clay used in the ceramic industry often contains iron oxide. In order to remove iron oxide, clay and water can be mixed together to form a suspension. Because clay particles are negatively charged and iron oxide particles are positively charged, very pure clay will gather near the anode after being electrified. Electrophoresis is also used for dust removal in factories. Electrophoresis can also detect the separated substances and play an important role in biochemical and clinical diagnosis. From the late 1940s to the early 1950s, electrophoresis using supports, such as filter paper electrophoresis, cellulose acetate membrane electrophoresis and agar electrophoresis, was developed one after another. In the late 1950s, starch gel electrophoresis and polyacrylamide gel electrophoresis appeared again. Applied electrophoresis has been widely used in analytical chemistry, biochemistry, clinical chemistry, toxicology, pharmacology, immunology, microbiology, food chemistry and other fields. In DC electric field, the phenomenon that charged particles move to electrodes with opposite signs is called electrophoretic resistance. 1807, Ferdinand Frederic Rheus of Moscow University first discovered electrophoresis, but it was not until 1937 that Thyselius of Sweden established boundary electrophoresis for separating protein that electrophoresis technology began to be applied. In 1960s and 1970s, electrophoresis technology developed rapidly after media such as filter paper and polyacrylamide gel were introduced into electrophoresis. The colorful electrophoresis forms make it widely used. Electrophoresis is not only used for the separation and analysis of small molecules, but also for the study of protein, nucleic acids, enzymes and even viruses and cells. Some electrophoresis methods have become common techniques in medical examination because of their simple equipment, convenient operation, high resolution and selectivity. Electroforming electrophoresis is also called electrocoating, electrophoretic paint and electrodeposition. Founded in 1960s, it was first used as automobile primer by Ford Motor Company. Because of its excellent anti-corrosion and anti-rust function, it was quickly widely used in military industry. In recent years, it has been applied to the surface treatment of daily hardware. Because of its excellent quality and high environmental protection, it is gradually replacing the traditional painting. Electrophoretic paint film Electrophoretic paint film has the advantages of full, uniform, flat and smooth coating. The hardness, adhesion, corrosion resistance, impact resistance and permeability of electrophoretic paint film are obviously superior to other coating processes. Detailed features: (1) Water-soluble coating is adopted, and water is used as the dissolution medium, which saves a lot of organic solvents, greatly reduces air pollution and environmental hazards, is safe and hygienic, and avoids fire hazards; (2) The coating efficiency is high, the coating loss is small, and the coating utilization rate can reach 90% ~ 95%; (3) The film thickness is uniform, the adhesion is strong, and the coating quality is good, so that all parts of the workpiece, such as inner layers, depressions, welds, etc., can obtain uniform and smooth paint films. So as to solve the problem of coating complicated workpieces by other coating methods; (4) the production efficiency is high, and the construction can realize automatic continuous production, which greatly improves the labor efficiency; (5) The equipment is complex, the investment cost is high, the power consumption is high, the temperature required for drying and curing is high, the painting management is complex, the construction conditions are harsh, and wastewater treatment is needed; (6) Only water-soluble paint can be used, and the color cannot be changed during painting, so the stability of the paint is difficult to control after long-term storage. (7) Electrophoretic coating equipment is complex and high in technology, which is suitable for color fixing production. Electrophoretic species moving interface electrophoresis is to put the separated ion (such as anion) mixture at one end (such as negative electrode) of the electrophoresis tank, and the sample has a clear interface with the carrier electrolyte before electrophoresis. After the electrophoresis starts, the charged particles move to the other electrode (positive electrode), with the fastest swimming ion ahead, and other ions are arranged in the order of electrode speed to form different regions. Only the interface in the first region is clear and completely separated, which contains ions with the fastest electrophoresis speed, and most other regions overlap. Electrophoresis is to put the sample in the middle of a support in a uniform carrier electrolyte. Under the action of electric field, the positively or negatively charged ions in the sample move to the negative electrode or the positive electrode at different speeds and are separated into separate regions. According to the different physical properties of the support, zone electrophoresis can be divided into paper and other fiber membrane electrophoresis, powder electrophoresis, gel electrophoresis and silk thread electrophoresis. Isoelectric focusing electrophoresis is to add amphoteric electrolyte into an electrophoresis tank filled with pH gradient buffer. When it is in an environment below its isoelectric point, it will be positively charged and move to the negative electrode. If it is in an environment higher than its isoelectric point, it will move negatively to the positive electrode. When swimming to its unique isoelectric point, its net charge is zero, the swimming speed drops to zero, and the substances with different isoelectric points finally focus on its own isoelectric point, forming a clear area with high resolution. Isokinetic electrophoresis is to add precursor ions (whose mobility is greater than all separated ions) and terminal ions (whose mobility is less than all separated ions) to the sample, and the sample is added between the precursor ions and the terminal ions. Under the action of external electric field, each ion is moving, and after a period of electrophoresis, it is completely separated. The spectral bands of separated ions are arranged between the spectral bands of leading ions and terminal ions in turn according to mobility. Because no suitable supporting electrolyte is added to carry the current, the obtained zones are interconnected (Figure D), and the interface is clear due to the "self-correction" effect, which is different from zone electrophoresis. Schematic diagram of electrophoresis separation principle A moving interface electrophoresis B zone electrophoresis C isoelectric focusing electrophoresis D isokinetic electrophoresis L leading ion T terminal ion. Overview of electrophoresis principle electrophoresis is that charged coating ions move to the cathode under the action of voltage applied to the anode and cathode, and react with alkaline substances produced on the cathode surface to form insoluble substances, which are deposited on the workpiece surface. It includes four processes: electrolysis (decomposition) is the electrolytic reaction at the beginning of the cathode reaction, which produces hydrogen and hydroxyl ions, and this reaction forms a strongly alkaline boundary layer on the cathode surface. When cations react with hydroxyl groups to become water-insoluble substances, films are deposited, and the equation is: H2O → OH+ H. The process in which cationic resin and H+move to the cathode and anions move to the anode under the action of electric field. Electrodeposition (precipitation) occurs on the surface of the coated workpiece. Cationic resin reacts with the cathode surface in an alkaline way, neutralizes without precipitation and deposits on the coated workpiece. Electroosmosis (dehydration) coating solid and the coating film on the surface of the workpiece are translucent and have many pores. Water is discharged from the cathode coating, so that the coating is dehydrated under the action of electric field, and the coating is adsorbed on the surface of the workpiece, thus completing the whole electrophoresis process. Basic Principles Most biological macromolecules such as protein, nucleic acids and polysaccharides have cationic and anionic groups, which are called zwitterions. Usually dispersed in solution in the form of particles, their electrostatic charge depends on the concentration of H+ in the medium or the interaction with other macromolecules. In the electric field, charged particles migrate to the cathode or anode, and the migration direction depends on their charged symbols. This migration phenomenon is called electrophoresis. If the colloidal solution of biological macromolecules is placed in an undisturbed electric field, the driving force for the particles to have constant mobility comes from the effective charge Q and the potential gradient E on the particles. They compete with the friction resistance f of the medium. In the free solution, this kind of competition obeys Stokes' law. F=6πrvη where v is the moving speed of particles with radius r in a medium with viscosity η. But in gel, this competition does not completely conform to Stokes' law. F depends on other factors in the medium, such as gel thickness, particle size and even the penetration of the medium. Electrophoretic mobility m is defined as the migration distance of particles in time t under the influence of potential gradient e =-or m = v/e, and the difference of mobility provides the basis for separating substances from the mixture, and the migration distance is proportional to the mobility. Electrophoresis refers to the phenomenon that charged particles or molecules move in an electric field, which is called electrophoresis. Macromolecule protein, polypeptide, virus particle, even cell or small molecule amino acid, nucleoside, etc. Orienteering can be done in an electric field. Tiselius successfully developed an interface electrophoresis instrument for serum protein electrophoresis, which was carried out in the free solution of U-tube. After electrophoresis, the refractive index differences formed by various protein were made into curve images by optical system, and the serum albumin was divided into albumin. There are five kinds of α 1- globulin, α2- globulin, β-globulin and γ-globulin. Subsequently, Wielamd and Kanig were equal to 1948, and paper electrophoresis was successfully carried out with filter paper strips as carriers. Since then, electrophoresis technology has been gradually accepted and valued by people, and then developed into filter paper, various cellulose powders, starch gels, agar and agarose gels, cellulose acetate films, polyacrylamide gels and so on. As a carrier, it is combined with dye enhancers such as silver ammonia dyeing and Coomassie brilliant blue. , greatly improving and promoting the color development and resolution of biological samples. In addition, the combination of electrophoretic separation and immune reaction makes the resolution develop towards micro and ultra-micro (1 ng ~). This paper mainly introduces the general principle and application of general electrophoresis. Factors affecting related books 1. The pH value of the electrophoretic medium determines the dissociation degree of the charged substance, and also determines the amount of net charge carried by the substance. For protein, amino acids and other similar amphoteric electrolytes, the farther the pH value is from the isoelectric point, the more charges the particles carry and the faster the swimming speed, and vice versa. Therefore, when separating a mixture, we should choose a pH value that can amplify the difference of various protein charges, so as to facilitate the effective separation of various protein. In order to keep the pH value of the solution constant during electrophoresis, we must use buffer solution. The ionic strength of the buffer solution is 1/2 of the sum of the product of the molar concentration of each ion in the solution and the square of the ionic valence number. The mobility of charged particles is proportional to the square root of ionic strength. When the ionic strength is low, the mobility is fast, but the ionic strength is too low, and the buffer capacity of the buffer solution is small, so it is difficult to keep the pH constant. At high ionic strength, the mobility is slow, but the electrophoresis band is narrower than that at low ionic strength. Generally, the ionic strength of the solution is between 0.02 and 0.2. I= 1/2∑CiZi2 (I: ionic strength; Ci: molar concentration of ions; Son: ionic valence. The ionic strength of 0. 154M NaCl solution is: I =1/2 (0.154x12+0.154x12) = 0./. 2 (0.015× 2×12+0.015× 22) = 0.045 3. Electric field strength (electric field strength) refers to the potential drop (potential difference or potential gradient) per centimeter. The electric field intensity has a direct influence on the electrophoresis speed. The higher the electric field intensity, the more charged particles. According to the needs of the experiment, electrophoresis can be divided into two types: one is high-voltage electrophoresis, and the voltage used is 500 ~ 1000 V or higher. Because of the high voltage and short electrophoresis time (some samples need several minutes), it is suitable for the separation of low molecular weight compounds, such as amino acids and inorganic ions, including partial focusing electrophoresis separation and sequential electrophoresis separation. Because of the high voltage and large heat output, it is necessary to install a cooling device, otherwise the heat will denature protein and other substances, which cannot be separated. The excessive evaporation of water in the buffer solution will increase the ionic strength on the support (filter paper, film or gel, etc.). ), and the siphon phenomenon (the liquid in the electrophoresis tank is sucked to the support) will affect the separation of substances. The other is atmospheric electrophoresis, with low calorific value and room temperature of 65438. 4. Electroosmosis The movement of a liquid relative to a solid stationary phase in an electric field is called electroosmosis. In carrier electrophoresis, an important factor affecting electrophoresis movement is electroosmosis. The most common situation is that γ -globulin moves from the origin to the negative electrode, which is the reverse movement phenomenon caused by electroosmosis. The reason of electroosmosis is that the carrier often contains ionizable groups. For example, the filter paper contains hydroxyl groups and is negatively charged, and the aqueous solution in contact with the filter paper is positively charged, so the liquid moves to the negative electrode. Because electroosmosis often exists at the same time as electrophoresis, the moving distance of charged particles is also affected by electroosmosis. If the electrophoresis direction is opposite to the electroosmosis direction, the actual electrophoresis distance is equal to the electrophoresis distance plus the electroosmosis distance. Agar contains agar pectin and more sulfate, so the electroosmosis phenomenon is obvious during agar electrophoresis, and many globulins move to the negative electrode. When agarose after removing agar pectin is used in gel electrophoresis, the electroosmosis effect is greatly weakened, and the moving distance caused by electroosmosis can be observed with uncharged colored dye or colored dextran in the center of the support. Classification of electrophoresis The electrophoresis methods currently used can be roughly divided into three categories: micro-electrophoresis, free-interface electrophoresis and zone electrophoresis. Zone electrophoresis is widely used and can be divided into the following categories: according to the physical properties of the support, zone electrophoresis can be divided into: (1) paper electrophoresis with filter paper as the support; (2) Powder electrophoresis: such as cellulose powder, starch and glass powder electrophoresis; (3) Gel electrophoresis: such as agar, agarose, silica gel, starch gel and polyacrylamide gel electrophoresis; (4) Edge line electrophoresis: for example, nylon and rayon electrophoresis. 2. According to the different forms of scaffolds, zone electrophoresis can be divided into: (1) plate electrophoresis: the scaffold is placed horizontally, which is the most commonly used electrophoresis method; (2) Vertical plate electrophoresis: polyacrylamide gel can be made into vertical plate electrophoresis. (3) Column (tube) electrophoresis: Polyacrylamide gel can be poured into a suitable electrophoresis tube for tube electrophoresis. 3. According to different pH continuity, zone electrophoresis can be divided into: (1) continuous pH electrophoresis, such as paper electrophoresis and cellulose acetate film electrophoresis; (2) Discontinuous pH electrophoresis: such as polyacrylamide gel disc electrophoresis; Measuring instruments The instruments needed for electrophoresis are: electrophoresis tank and power supply. 1. Electrophoresis bath Electrophoresis bath is the core part of electrophoresis system. According to the electrophoresis principle, an electrophoresis bracket is placed between two buffers, and an electric field is connected with the two buffers through the electrophoresis bracket, and different electrophoresis tanks are used for different electrophoresis. Commonly used electrophoresis tanks are: (1) disc electrophoresis tank: there are two electrophoresis tanks and a cover with platinum electrode. There are several holes in the water tank above. When these holes are not used, they should be plugged with silicone rubber plugs. The hole to be used should be equipped with a silicone rubber plug, and an electrophoresis tube (glass tube) can be inserted. The inner diameter of electrophoresis tube was 5 ~ 7 mm in the early days, but now it is getting thinner and thinner to ensure heat dissipation and miniaturization. (2) Vertical plate electrophoresis tank: The basic principle and structure of vertical plate electrophoresis tank are basically the same as that of disk electrophoresis tank. The only difference is that the glue making and electrophoresis are not in the electrophoresis tube, but in the middle of a vertically placed parallel glass plate. (3) Horizontal electrophoresis tank: Horizontal electrophoresis tanks have different shapes, but basically the same structure. Generally, it comprises an electrophoresis tank base, a cooling plate and electrodes. In order to make charged biological macromolecules swim in the electric field, an electric field must be applied, and the resolution and speed of electrophoresis are closely related to the electrical parameters during electrophoresis. Different electrophoresis technologies need different voltage, current and power ranges, so the choice of power supply mainly depends on the needs of electrophoresis technologies, such as polyacrylamide gel electrophoresis and SDS electrophoresis, which require a voltage of 200 ~ 600 V. Apply 1. Polyacrylamide gel electrophoresis can be used to identify the purity of protein. Polyacrylamide gel electrophoresis has both charge effect and molecular sieve effect, which can separate substances with the same molecular size but different molecular sizes. Its resolution is much higher than that of ordinary chromatography and electrophoresis methods, and it can detect samples of10-9 ~10-12g with good repeatability and no electroosmosis. 2. The molecular weight of protein can be determined by 2.SDS polyacrylamide gel electrophoresis. The principle is that SDS with a large amount of charge is combined with protein molecule to overcome the influence of the original charge of protein molecule and obtain a constant charge-mass ratio. SDS polyacrylamide gel electrophoresis has been successfully used to determine the molecular weight of protein. This method has the advantages of short determination time, high resolution and small sample size (1 ~ 100μ g), but it is only suitable for spherical or basically spherical protein. Some protein are not easy to combine with SDS, such as papain and ribonuclease. At this time, the determination result is inaccurate. Electrophoresis tube 3. Polyacrylamide gel electrophoresis can be used for protein quantification. The gel after electrophoresis is scanned by gel scanner, which is mainly used to scan the strips after one-dimensional electrophoresis and the spots after two-dimensional electrophoresis. 4. Agar or agarose gel immunoelectrophoresis can be used to check the purity of protein preparation; (2) analyzing the composition of protein mixture; ③ To study whether there are antibodies against a known antigen in antiserum preparation; ④ Check whether the two antigens are the same. Results Different methods should be used for different purposes. The combination of dyes and biological macromolecules to form colored complexes is the most commonly used method for post-electrophoresis detection. (VII) Abnormal results of polyacrylamide gel electrophoresis and countermeasures 1. The leading edge of the indicator appears up or down. The upward "smile" phenomenon shows that the gel is not cooled evenly, and the middle part is not cooled well, which leads to different mobility of molecules in the gel. This often happens in thick gel and vertical electrophoresis. The downward "frown" phenomenon is often caused by improper installation of electrophoresis tank during vertical electrophoresis, especially when there are bubbles at the bottom of the "sandwich" composed of gel and glass plate or the gel polymerization near the spacer is incomplete. 2. "Tailing" is the most common phenomenon in electrophoresis. This is usually caused by poor dissolution of the sample. The solution is to centrifuge before adding samples, select appropriate sample buffer and gel buffer, and add solubilizing auxiliary reagents. Another method is to reduce the gel concentration. 3. The phenomenon of "texture" is often caused by insoluble particles in the sample, and the way to overcome it is to increase solubility and remove insoluble particles by centrifugation. 4. The shift of protein band is often caused by the non-parallel placement of filter paper strips or electrodes, or the shift of injection position. 5. The protein band is too wide, which is connected with the protein band adjacent to the protein lane, which is caused by too many samples or leakage of the sample hole. 6. The unclear protein stripe and poor resolution are caused by many reasons. Although gradient gel can improve the resolution, compared with other methods, conventional polyacrylamide gel electrophoresis is a method with lower resolution. To improve the resolution, don't add too many samples. Small samples will produce narrow bands. Electrophoresis should be carried out immediately after sample addition to prevent diffusion. Choosing appropriate gel concentration can make the components fully separated. Usually, the resolution of the protein band near the front end is not good. It is necessary to pour enough gel according to the relationship between molecular weight and gel pore size so that the sample will not go out of the front end. The proteolysis of the sample will also cause diffusion and reduce the resolution. Hydrolysis usually occurs during sample preparation, and the endogenous protease in the system will hydrolyze the sample protein. This situation can be reduced if protease inhibitors are added to the buffer.