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Electrophoresis is a phenomenon in which charged particles move in an electric field toward an electrode that has an opposite charge to themselves. Different charged particles swim at different speeds in the same electric field. It is often expressed in terms of mobility (or mobility). The mobility is the speed of electrophoresis of charged particles per unit electric field strength. Electrophoresis techniques are divided into paper electrophoresis, cellulose acetate film electrophoresis, starch gel electrophoresis, agar (sugar) gel electrophoresis and polyacrylamide gel electrophoresis by the support. The shape of the gel is divided into horizontal plate electrophoresis, garden plate column electrophoresis and vertical plate electrophoresis.
Electrophoresis with supporting body:
1. Paper electrophoresis
2. Acetate film electrophoresis
3. Thin-layer electrophoresis
4. Non-gel supporting body zone electrophoresis (Supporting body: starch, cellulose powder, glass powder, silica gel, etc.)
5. Gel supporting body zone electrophoresis ① Starch liquid ② Polyacrylamide gel ③ Agar (sugar) gel ③ Agar (sugar) gel electrophoresis ③ Agar (sugar) gel electrophoresis ① Polyacrylamide gel electrophoresis ② Polyacrylamide gel electrophoresis ③ Agar (sugar) gel electrophoresis ③ Polyacrylamide gel electrophoresis Gel ③ Agar (sugar) gel
Electrophoresis techniques without support body:
1. Tiselius or micro electrophoresis
2. Micro electrophoresis
3. Isoelectrode focusing electrophoresis technique
4. Isovelocity electrophoresis technique
5. Density gradient electrophoresis
Electrophoresis applications
Application of electrophoresis techniques
In a direct current electric field, the phenomenon of charged particles moving to electrodes with opposite sign is called electrophoresis (electropho-resis). 1809 Russian physicist Peнce first discovered the phenomenon of electrophoresis, but it was not until 1937 that Sweden's Tiselius established interface electrophoresis (boundary) for separating proteins. electrophoresis (boundary electrophoresis) for separating proteins was established by Tiselius in Sweden in 1937. In the 60s and 70s of this century, when filter paper, polyacrylamide gel and other media were successively introduced into electrophoresis, electrophoresis technology was rapidly developed. The colorful forms of electrophoresis make it widely used. In addition to the separation and analysis of small molecules, electrophoresis is mainly used for the study of proteins, nucleic acids, enzymes, and even viruses and cells. Due to some electrophoresis method equipment is simple, easy to operate, with high resolution and selectivity characteristics, has become a commonly used technology in medical testing.
First, the basic principles of electrophoresis technology and classification
In the electric field, to promote the movement of the charged point of force (F) is equal to the net charge of the point of charge (Q) and the product of electric field strength (E).
F = QE
The forward movement of the mass is also affected by the resistance (F), for a spherical mass, obeys Stoke's law, that is:
F′ = 6πrην
where r is the radius of the mass, η is the viscosity of the medium, ν is the speed of movement of the mass, when the mass is moving steadily in the electric field:
F = F′ That is, QE = 6πrην
The exchange of the above formula can be written as
ν/E means the unit electric field strength of the speed of movement, can be used in the mobility of μ, that is,
From the above formula, the mobility of the spherical particle, first of all depends on the state of its own, that is, with the amount of charged proportional to the radius of the medium and its viscosity inversely proportional to. In addition to its own state of factors, other factors in the electrophoresis system also affect the electrophoretic mobility of the plasma point.
Electrophoresis can be divided into two categories: free electrophoresis (without support) and zone electrophoresis (with support). The former includes Tise-leas type microelectrophoresis, microelectrophoresis, isoelectric focusing electrophoresis, isokinetic electrophoresis and density gradient electrophoresis. Zone electrophoresis, on the other hand, includes filter paper electrophoresis (atmospheric pressure and high pressure), thin-layer electrophoresis (film and sheet), and gel electrophoresis (agar, agarose, starch gel, polyacrylamide gel).
The development of free electrophoresis method is not rapid, because its electrophoresis instrument construction is complex, large volume, strict operation requirements, expensive, etc.. Zone electrophoresis can be used with various types of substances as a support body, and its application is more extensive. In this section, only a few commonly used zone electrophoresis are described.
Second, the factors affecting electrophoretic mobility
⒈ electric field strength The electric field strength refers to the unit length (cm) of the potential drop, also known as the potential gradient. If the filter paper as a support, its ends are immersed in the electrode liquid, electrode liquid and filter paper interface of the paper length of 20cm, the measured potential drop of 200V, then the electric field strength of 200V/20cm = 10V/cm. when the voltage is 500V or less, the electric field strength of 2-10v/cm for atmospheric pressure electrophoresis. When the voltage is above 500V and the electric field strength is 20-200V/cm, it is high voltage electrophoresis. The electric field strength is large, and the mobility of charged plasmas is accelerated, so it saves time, but because it generates a large amount of heat, it should be equipped with a cooling device to maintain constant temperature.
⒉pH value of the solution pH of the solution to determine the degree of dissociation of the separated substances and the charged nature of the plasma point and the amount of net charge. For example, the protein molecule, it is both acidic groups (-COOH), and basic groups (-NH2) of the amphoteric electrolyte, in a solution with equal positive and negative charges, that is, the net charge of the molecule is equal to zero, at this time, the protein is no longer moving in the electric field, the pH of the solution for the protein isoelectric point (isoelctric point, pI). If the pH of the solution is on the acid side of the isoelctric point, i.e., pH < pl, the protein is positively charged and moves toward the negative pole in the electric field. If the solution pH is on the alkaline side of the isoelectric point, i.e., pH > pl, the protein is negatively charged and moves toward the positive pole. The farther the pH of the solution is from pl, the more net charge the plasmid carries and the greater the electrophoretic mobility. Therefore, in electrophoresis, according to the nature of the sample, should choose the appropriate pH buffer.
兾3 solution of ionic strength electrophoresis solution ion concentration increases will cause a decrease in the mobility of the plasma point. The reason is that the charged plasma attracts the opposite conformity of the ions gathered around it, the formation of a plasma with the movement of the opposite conformity of the ionic atmosphere (ionic atmosphere), the ionic atmosphere not only reduces the amount of charged plasma point, while increasing the resistance of the plasma point to move forward, and even to make it can not be swum. However, too low a concentration of ions will reduce the total concentration of buffer and buffer capacity, not easy to maintain the pH value of the solution, affecting the amount of charged plasma point, changing the speed of swimming. This barrier effect of ions is related to their concentration and valence. It can be expressed as ionic strength I.
Where S represents the ionic species in solution, and Ci and Zi represent the molar concentration and valence of each ion, respectively. The most common values of I are between 0.02 and 0.2.
Singed electroosmosisThe relative movement of a liquid against a solid support under an electric field is called electro-o *** osis. The reason for this is that the solid support porous, and with dissociable chemical groups, so often adsorbed in the solution of positive or negative ions, so that the solution is relatively negatively or positively charged. Such as filter paper as a support, paper cellulose adsorption OH- negatively charged, and paper contact with the aqueous solution due to the production of H3O+, positively charged to move to the negative pole, if the mass of the original in the electric field to move to the negative pole, the result of the mass of the performance of the velocity than its intrinsic speed, if the mass of the original to move to the positive, the performance of the velocity than its intrinsic speed to be slower, it can be seen that as far as possible to select the role of the support of the low-permeability in order to minimize the impact of electroosmosis
Three.
Third, electrophoretic analysis of commonly used methods
(a) cellulose acetate film electrophoresis
Cellulose acetate is the formation of cellulose acetate acetylation of cellulose hydroxyl. The film made of this substance is called cellulose acetate film. This film on the protein sample adsorption is small, almost completely eliminate the paper electrophoresis in the "tail" phenomenon, and because the membrane hydrophilicity is relatively small, it holds less buffer, electrophoresis, most of the current conducted by the sample, so the separation speed is fast, the electrophoresis time is short, the sample dosage is small, 5 μg of protein can be obtained to satisfy the separation effect. Therefore, the separation speed is fast, the electrophoresis time is short, and the sample dosage is small. Therefore, it is especially suitable for the detection of abnormal protein in pathological conditions.
The cellulose acetate membrane is treated with ethanol glacial acetic acid or other transparent solutions to make the membrane transparent, which is conducive to the determination of light absorption scanning of electrophoretic patterns and long-term preservation of the membrane.
⒈ materials and reagents ⒈ cellulose acetate membrane is generally used commercially available products, commonly used electrophoresis buffer pH8.6 barbiturate buffer, concentration in 0.05-0.09mol/L.
⒉操作要点
⑴ membrane pretreatment: must be in the buffer before electrophoresis will be soaked in buffer, soaked through the membrane, remove the membrane and absorb excess buffer with filter paper, do not absorb too dry. The membrane must be soaked in the buffer solution before electrophoresis.
(2) Sample addition: the amount of sample depends on the concentration of the sample, its nature, staining methods and detection methods and other factors. Routine electrophoretic analysis of serum proteins, not more than 1μl per cm spiked line, equivalent to 60-80μg of protein.
⑶ Electrophoresis: can be performed at room temperature. The voltage is 25V/cm and the current is 0.4-0.6mA/cm wide.
(4) Staining: general protein staining is often used amino black and Li Chun red, glycoproteins with toluidine blue or periodic acid-Schiff reagent, lipoproteins with Sudan black or magenta sulfite staining.
(5) decolorization and transparency: the most commonly applied decolorizing agent for water-soluble dyes is 5% aqueous acetic acid. For long term storage or light absorption scanning measurement, it can be immersed in a transparent solution of glacial acetic acid: anhydrous ethanol = 30:70 (V/V).
(ii) Gel electrophoresis
The zone electrophoresis method using starch gel, agar or agarose gel, polyacrylamide gel, etc., as support medium is called gel electrophoresis. Among them, polyacrylamide gel electrophoresis (polyacrylamide gel electrophoresis, PAGE) is commonly used to separate proteins and smaller molecules of nucleic acids. Agarose gel pore size is large, the general protein does not play a role in molecular sieving, but is suitable for the separation of isoenzymes and their isoforms, large molecules of nucleic acids and other widely used, are introduced as follows:
⒈ agarose gel electrophoresis principle is summarized in agarose is separated from agar preparation of chain polysaccharides. Its structural units are D-galactose and 3.6-anhydro-L-galactose. Many agarose chains are coiled around each other by hydrogen bonding and other forces to form rope-like agarose bundles, constituting a large mesh-type gel. Therefore, this gel is suitable for the separation, identification and purification of immune complexes, nucleic acids and *** white. It is often used for the detection of isoenzymes such as LDH and CK in clinical biochemical tests.
⒉Agarose gel electrophoresis separation of nucleic acids in a certain concentration of agarose gel medium, the electrophoretic mobility of DNA molecules and its molecular weight of the common logarithm of the inversely proportional; molecular conformation also has an impact on the mobility, such as *** valence of the closed-loop DNA> linear DNA> open-loop double-stranded DNA. when the concentration of the gel is too high, the pore size of the gel becomes smaller, the ring of DNA (spherical) cannot Enter the gel, the relative mobility is 0, while the same size of linear DNA (rigid rod) can move forward in the direction of the long axis, the relative mobility is greater than 0.
(1) equipment and reagents: agarose gel electrophoresis is divided into two types of vertical and horizontal type. Among them, the horizontal type can prepare low concentration agarose gel, and the preparation of gel and adding samples are more convenient, so it is more widely used. Nucleic acid separation generally use continuous buffer system, commonly used TBE (0.08mol/l Tris-HCl, pH8.5, 0.08mol/L boric acid, 0.0024mol/l EDTA) and THE (0.04mol/l Tris-HCl. pH7.8, 0.2mol/L sodium acetate, 0.0018mol/l EDTA). ).
(2) Gel preparation: 0.5%-0.8% agarose gel solution was prepared with the above buffer, heated in a boiling water bath or microwave oven to melt it, and then ethidium bromide (EB) was added to the final concentration of 0.5 μg/ml when it was cooled down to 55 ℃, and then injected into the assembled molds of glass or plexiglass plates, with the thickness according to the concentration of the samples. When injecting the glue, the lower end of the comb teeth is 0.5-1.0mm from the glass plate, and when it is de-solidified, the comb is removed, and an appropriate amount of electrode buffer is added so that the plate glue is submerged 1mm below the buffer.
(3) sample preparation and addition: dissolved in TBE or THE sample should contain indicator dye (0.025% bromophenol blue or orange-yellow orange), sucrose (10%-15%) or glycerol (5%-10%), you can also use 2.5% Fico Ⅱ to increase the specific gravity of the sample, so that the sample is concentrated, each hole can be added to the sample of 5-10 μg.
(4) electrophoresis: the general voltage of 5-15V/cm. For the separation of large molecules, the voltage can be 5V/cm. the electrophoresis process is best carried out under low-temperature conditions.
(5) Sample recovery: observe the separation of samples under the UV lamp after the end of electrophoresis, the DNA molecules or special fragments needed can be recovered from the gel after electrophoresis by different methods, such as electrophoresis elution method: cut the gel containing nucleic acid zone bands under the UV lamp, put it into the dialysis bag (which contains an appropriate amount of fresh electrophoresis buffer), tie the bag tightly and then flatly put it in the shallow buffer between two electrodes in the horizontal electrophoresis bath, and then put it in the shallow buffer between two electrodes in the horizontal electrophoresis tank. After tightening the dialysis bag, put it flatly in the shallow buffer between two electrodes of the horizontal electrophoresis tank, electrophoresis at 100V for 2-3 hours, and then exchange the positive and negative electrodes and electrophoresis in the reverse direction for 2 minutes, so that the DNA on the dialysis bag can be released. The DNA-containing solution is aspirated, and phenol extraction and ethanol precipitation steps are performed to complete the sample recovery.
There are other methods such as low melting point agarose method, ammonium acetate solution leaching method, frozen extrusion method, etc., but all methods are only conducive to the recovery of small molecular weight DNA fragments (<1kb), and as the molecular weight of the DNA increases, the amount of recovery decreases significantly.
(C) Isoelectric focusing electrophoresis
Isoelectric focusing (IEF) is an electrophoretic technique introduced in the mid-1960s that utilizes a medium with a pH gradient to separate proteins with different isoelectric points. Because of its resolution of up to 0.01 pH units, it is particularly suitable for the separation of protein components with similar molecular weights but different isoelectric points.
⒈ IEF basic principles in the electrophoresis of IEF, with a pH gradient of the medium its distribution is from the anode to the cathode, the pH value gradually increases. As mentioned earlier, the protein molecules have the characteristics of amphoteric dissociation and isoelectric point, so that the protein molecules in the alkaline region with a negative charge to the anode to move until a certain pH point when the loss of charge and stop moving, the pH of the medium here is exactly equal to the focus of the protein molecules of the isoelectric point (pl). Similarly, protein molecules located in the acidic region move toward the cathode with a positive charge until they focus on their isoelectric point. It can be seen in the method, the isoelectric point is a measure of the properties of the protein components, the isoelectric point of different protein mixtures will be added to the gel medium with a pH gradient, in the electric field after a certain period of time, the components will be focused on the respective isoelectric point of the corresponding pH position, the formation of separated protein zones.
⒉pH gradient composition of the composition of the pH gradient there are two ways, one is the artificial pH gradient, due to its instability, poor reproducibility, is no longer used. The other is a natural pH gradient. The establishment of natural pH gradient is to introduce a series of mixtures of amphoteric electrolytes with isoelectric points close to each other between the positive and negative poles of a horizontal plate or an electrophoresis tube, and to inhale acids such as sulfuric acid, phosphoric acid, or acetic acid, etc., at the positive pole, and to introduce bases such as sodium hydroxide and ammonia, etc., at the negative pole. Before electrophoresis begins, the pH of the mixture of the two sexes electrolytes is a mean value, that is, the pH in each section of the medium is equal, expressed by pH0. After the start of electrophoresis, the molecule with the lowest pH in the mixture has the most negative charge, pI1 is its isoelectric point, and it moves the fastest to the positive pole, and when it moves to the interface of the acid-liquid near the positive pole, the pH suddenly decreases, and it is even close to or a little bit lower than that of PI1, and this molecule no longer moves forward and stays in this region. As the amphoteric electrolyte has a certain buffering capacity, so that the pH of the medium in a certain region around it to maintain in its isoelectric point range. pH is slightly higher than the second amphoteric electrolyte, its isoelectric point of pI2, also moved to the positive pole, due to the pI2> pI1, so it is located in the first amphoteric electrolyte after the electrolyte, so after a certain period of time, the amphoteric electrolytes with different isoelectric point according to their respective isoelectric point In this way, after a certain period of time, the amphoteric electrolytes with different isoelectric points are arranged sequentially, forming a linear pH gradient from positive to negative isoelectric point increasing from low to high, as shown in Figure 16-3.
Amphoteric electrolyte carrier and support medium Ideal amphoteric electrolyte carrier should have sufficient buffering capacity and conductivity at pI, the former to ensure the stability of the pH gradient, the latter allows a certain amount of current through. Amphoteric electrolytes with different pI should have similar conductivity coefficients so that the conductivity of the whole system is uniform. The molecular weight of the amphoteric electrolyte should be small, so that it is easy to separate it from the separated macromolecular substances by molecular sieve or dialysis method, and it should not react with or denature the separated substances.
Figure 16-3 Schematic diagram of pH gradient formation
Commonly used pH gradient support media are polyacrylamide gel, agarose gel, dextran gel, etc., of which polyacrylamide gel is the most commonly used.
After electrophoresis, the gel should not be stained directly with stains because commonly used protein stains can also bind to amphoteric electrolytes, so the gel should be soaked in 5% trichloroacetic acid to remove the amphoteric electrolytes before staining it in an appropriate way.
(D) other electrophoresis techniques
⒈ IEF / SDS-PAGE bidirectional electrophoresis 1975 O′Farrall et al. According to the isoelectric point difference between different components and molecular weight difference established IEF / Sd S-PAGE bidirectional electrophoresis. Where IEF electrophoresis (tube column) is the first direction and SDS-PAGE is the second direction (plate). In the first direction of IEF electrophoresis, the electrophoresis system should be added a high concentration of urea, appropriate amount of non-ionic detergent NP-40. protein samples should contain these two substances in addition to dithiothreitol to promote protein denaturation and peptide chain stretching.
At the end of IEF electrophoresis, the cylindrical gel is equilibrated by oscillation in the sample treatment solution (containing SDS, β-mercaptoethanol) applied for SDS-PAGE, and then embedded in the upper end of the gel plate of SDS-PAGE, which is ready to be subjected to the second direction electrophoresis.
The separation of proteins (including ribosomal proteins, histones, etc.) by IEF/ SDS-PAGE bidirectional electrophoresis is extremely fine, making it particularly suitable for separating complex protein fractions in bacteria or cells.
⒉Capillary electrophoresis Neuhoff et al. in 1973 established the capillary uniform concentration and gradient concentration gel used to analyze trace proteins, that is, microcolumn gel electrophoresis, uniform concentration of the gel is the capillary immersed in the gel mixture, so that the gel filled with about 2/3 of the total volume, and then snapped into a substitute clay pad of about 2mm thick, closed the bottom of the tube with a diameter The bottom of the tube was closed, and a rigid glass capillary tube with a diameter thinner than that of the capillary tube holding the gel was used to absorb the water and spread it over the gel. After polymerization, remove the water layer and add protein solution (0.1-1.0 μl, concentration of 1-3 mg/ml) to the gel with the capillary, fill the gap of the capillary with electrode buffer, excise the insertion of the clay part of the electrophoresis.
The birth of the capillary electrophoresis analyzer, especially the U.S. Biosystems high-performance electrophoresis chromatography for DNA fragments, proteins and peptides, and other biological macromolecules, the separation and recovery of a rapid and effective way. High performance electrophoresis chromatography is a combination of gel electrophoresis resolution and fast liquid chromatography. When eluting samples from a gel, a continuous stream of eluent carries the separated components, and the results are displayed and printed for record by an on-line detector. High-performance electrophoresis chromatography has the advantages of high resolution and biocompatibility inherent in gel electrophoresis, and the convenience of continuous sample elution.