(a) Cellulose acetate membrane electrophoresis Cellulose acetate is a cellulose acetate formed by hydroxyacetylation of cellulose. The film made of this substance is called a cellulose acetate film. This kind of film has low adsorption to protein samples and can almost completely eliminate the "tailing" phenomenon in paper electrophoresis. Because the hydrophilicity of the membrane is relatively small, it also contains less buffer, and most of the current during electrophoresis. Since the sample is conducted, the separation speed is fast, the electrophoresis time is short, and the amount of sample is small. A 5 μg protein can be satisfactorily separated. Therefore, it is particularly suitable for the detection of trace abnormal proteins under pathological conditions. (b) Gel Electrophoresis Band electrophoresis using starch gel, agar or agarose gel, polyacrylamide gel, etc. as a supporting medium is called gel electrophoresis. Among them, polyacrylamide gel electrophoresis (PAGE) is widely used to separate proteins and smaller molecules of nucleic acids. Agarose gel has a large pore size and does not function as a molecular sieve for general proteins, but is suitable for the separation of isoenzymes and their isoforms. Macromolecular nucleic acids and other applications are widely used and are described as follows: Each electrophoresis method was operated as described below unless otherwise specified. Third, agarose gel electrophoresis Automatic 3D Fiber Laser Pipe Cutting Machine
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The cellulose acetate film can be transparent after treatment with glacial acetic acid ethanol solution or other transparent liquid, which facilitates the light absorption scanning measurement of the electrophoretic pattern and the long-term preservation of the film.
1. Materials and reagents Cellulose acetate membranes are generally commercially available. The commonly used electrophoresis buffer is a barbital buffer with a pH of 8.6 at a concentration of 0.05-0.09 mol/L.
2. Operation points (1) Pretreatment of the membrane: The membrane must be soaked in the buffer before electrophoresis. After soaking, remove the membrane and use a filter paper to remove excess buffer. Do not breathe too much dry.
(2) Sample addition: The amount of sample is determined by factors such as the sample concentration, the nature of the sample, the dyeing method, and the detection method. Conventional electrophoretic analysis of serum proteins does not exceed 1 μl per cm of sample line, corresponding to 60-80 μg of protein.
(3) Electrophoresis: It can be performed at room temperature. The voltage is 25V/cm and the current is 0.4-0.6mA/cm wide.
(4) Staining: Amino black and ponceau are often used for general protein staining, toluidine blue or periodic acid-Schiff reagent for glycoproteins, and Sudan black or fuchsin sulfuric acid for lipoproteins.
(5) Decoloration and transparency: The most commonly used decolorant for water-soluble dyes is a 5% aqueous acetic acid solution. For long-term preservation or light absorption scanning measurements, immersed in a transparent solution of glacial acetic acid: absolute ethanol = 30:70 (V/V).
1. Principle of agarose gel electrophoresis Overview Agarose is a chain polysaccharide prepared from agar. Its structural units are D-galactose and 3.6-anhydro-L-galactose. Many agarose chains are coiled around each other by hydrogen bonds and other forces to form a rope-like agarose bundle, forming a large mesh gel. Therefore, the gel is suitable for the separation, identification and purification of immune complexes, nucleic acids and nuclear proteins. In clinical biochemical tests are commonly used in the detection of LDH, CK and other workers.
2. Basic techniques for separation of nucleic acids by agarose gel electrophoresis In certain concentrations of agarose gel media, the electrophoretic mobility of DNA molecules is inversely proportional to the common logarithm of their molecular weight; the molecular configuration also has an effect on the mobility, such as covalent Closed-loop DNA> Straight-line DNA> Open-loop double-stranded DNA. When the gel concentration is too high, the pore size of the gel becomes smaller, circular DNA (spherical) cannot enter the gel, the relative mobility is 0, and the same size of linear DNA
(Rigid rods) can be advanced in the direction of the long axis with a relative mobility greater than zero.
(1) Equipment and reagents: Agarose gel electrophoresis is divided into vertical and horizontal types. Among them, the low-concentration agarose gel can be prepared in the horizontal type, and it is more convenient to make and add the sample. Therefore, the application is more extensive. Nucleic acid is generally separated by a 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, 0.2 mol/L sodium acetate, 0.0018 mol/L EDTA).
(2) Gel preparation: Prepare a 0.5%-0.8% agarose gel solution with the above buffer and heat it in a boiling water bath or microwave oven. Add EB to a final concentration of 0.5μg/ml when cooled to 55°C. Ml, then inject it into a glass or plexiglass assembled mold, the thickness of which depends on the sample concentration. When gluing, the lower end of the comb teeth is 0.5-1.0mm away from the glass plate. After desolidification, remove the comb and add appropriate amount of electrode buffer to immerse the glue in the buffer under 1mm.
(3) Sample preparation and sample addition: Samples dissolved in TBE or THE should contain indicator dye (0.025% bromophenol blue or orange orange), sucrose (10%-15%) or glycerin (5%-10%), also The specific gravity can be increased using 2.5% Fico II to concentrate the sample and 5-10 μg can be added per well.
(4) Electrophoresis: The general voltage is 5-15V/cm. A voltage of 5 V/cm can be used for the separation of macromolecules. The electrophoresis process is preferably performed at a low temperature.
(5) Sample recovery: After the end of electrophoresis, observe the separation of the sample under the UV lamp. The required DNA molecules or specific fragments can be recovered from the gel after electrophoresis by different methods, such as the electrophoresis elution method: under the UV lamp. Cut the gel containing the nucleic acid zone, and place it in a dialysis bag (containing a suitable amount of fresh electrophoresis buffer), tighten the dialysis bag, and place it in the shallow buffer between the two electrodes of the horizontal electrophoresis cell, 100V. Electrophoresis was performed for 2-3 hours, then the positive and negative electrodes were exchanged, and the electrophoresis was carried out for 2 minutes to release the DNA on the dialysis bag. Aspirate the solution containing DNA, perform phenol extraction, ethanol precipitation and other steps to complete the recovery of the sample.
Other methods include 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), with the increase in the molecular weight of DNA, recovery Decreased significantly.
(C) isoelectric focusing electrophoresis isoelectric focusing (IEF) is an electrophoretic technique using a pH gradient medium to separate proteins with different isoelectric points in the mid-1960s. Due to its resolution of up to 0.01 pH units, it is particularly suitable for the separation of protein components with similar molecular weights and different isoelectric points.
1. The basic principle of IEF In the electrophoresis of IEF, the medium with a pH gradient is distributed from the anode to the cathode, and the pH value gradually increases. As mentioned earlier, protein molecules have the characteristics of amphipathic dissociation and isoelectric point, so that in the basic region, the protein molecules move negatively to the anode until they lose their charge at a pH site and stop moving. The pH of the medium is It is exactly equal to the isoelectric point (pl) of the focused protein molecule. Similarly, protein molecules located in acidic regions carry a positive charge toward the cathode until they are focused on their isoelectric point. It can be seen that in the method, the isoelectric point is a characteristic measure of the protein component, and a protein mixture with different isoelectric points is added to a gel medium with a pH gradient. After a certain time in the electric field, each component will be focused on The respective isoelectric point corresponding to the pH position forms an isolated protein band.
2. Composition of pH Gradient There are two kinds of composition of pH gradients. One is an artificial pH gradient. Because of its instability and poor repeatability, it is no longer used. The other is a natural pH gradient. The establishment of a natural pH gradient is to introduce a mixture of amphoteric electrolytes whose isoelectric points are close to each other between the positive and negative electrodes of a horizontal plate or electrophoresis tube. At the positive end, an acid solution such as sulfuric acid, phosphoric acid or acetic acid is inhaled, and alkali is introduced at the negative electrode end. Liquid, such as sodium hydroxide, ammonia, etc. The pH of the mixture of the amphoteric electrolytes prior to the start of electrophoresis is a mean value, ie, the pH is equal in each section of the medium, and is represented by pH0. After the electrophoresis begins, the lowest pH molecule in the mixture has the most negative charge, and pI1 is its isoelectric point. It moves to the positive electrode at the fastest speed. When it moves to the interface of the acid near the positive electrode, the pH suddenly drops, even close to or slightly lower. In PI1, this molecule no longer moves forward and stays in this area. Because the ampholyte has a certain buffering capacity, the pH of the medium in a certain area around it is kept in its isoelectric point range. The slightly higher pH of the second amphoteric electrolyte, its isoelectric point is pI2, also moves to the positive electrode, because pI2>pI1, so positioned after the first ampholyte, so that after a certain time, with different isoelectric point Amphoteric electrolytes are arranged in order according to their respective isoelectric points, forming a linear pH gradient from the positive electrode to the negative electrode, such as increasing, from low to high, as shown in Figure 16-3.
3. Amphoteric electrolyte carrier and support medium The ideal ampholyte carrier should have sufficient buffering capacity and conductance at pI. The former ensures the stability of the pH gradient and the latter allows a certain current to pass. The amphiphilic electrolytes with different pIs should have similar conductivity coefficients so that the conductance of the entire system is uniform. The molecular weight of the amphoteric electrolyte is small, and it is easy to separate it from the separated polymer by molecular sieve or dialysis method, and it should not react with or denature the separated material.
Commonly used pH gradient support media are polyacrylamide gels, agarose gels, dextran gels, etc. Polyacrylamide gels are the most commonly used.
After electrophoresis, it is not possible to directly stain with a dye, because the commonly used protein dyes can also be combined with the amphoteric electrolyte. Therefore, the gel should be first soaked in 5% trichloroacetic acid to remove the ampholyte, and then stained with an appropriate method.
(IV) Other electrophoresis techniques 1. IEF/SDS-PAGE two-dimensional electrophoresis 1975 O'Farrall et al established IEF/SD S-PAGE two-dimensional electrophoresis based on differences in isoelectric point and molecular weight differences between different components. Among them, IEF electrophoresis (tube column) is the first direction, and SDS-PAGE is the second direction (plate). When carrying out the first-direction IEF electrophoresis, high concentration urea should be added to the electrophoresis system, and a suitable amount of non-ionic detergent NP-40 should be added. Dithiothreitol should be added to the protein sample in addition to these two substances to promote protein denaturation and peptide stretch. After the IEF electrophoresis was completed, the cylindrical gel was equilibrated in a sample treatment solution (containing SDS and β-mercaptoethanol) applied to SDS-PAGE, and then embedded in the SDS-PAGE gel plate. Second electrophoresis.
IEF/SDS-PAGE two-dimensional electrophoresis is extremely delicate for the separation of proteins (including ribosomal proteins, histones, etc.) and is therefore particularly suitable for the isolation of complex protein components in bacteria or cells.
2. Capillary electrophoresis Neuhoff et al. established in 1973 a method for the analysis of microproteins using capillary homogenization and gradient gels, ie microcolumn gel electrophoresis. A uniform concentration of gel is obtained by immersing the capillary in a gel mixture and allowing coagulation. The gel fills about 2/3 of the total volume and is then plunged into a surrogate clay pad about 2 mm thick. The bottom of the tube is closed and the gel is filled with a hard glass capillary tube with a diameter smaller than the capillary holding the gel. . After the polymerization, the aqueous layer was removed and a protein solution (0.1-1.0 μl, concentration of 1-3 mg/ml) was added to the gel with a capillary. The gaps in the capillaries were filled with the electrode buffer, and the portion of the clay was removed for electrophoresis.
The birth of the current capillary electrophoresis analyzer, in particular, the United States Biosystems high-performance electrophoresis chromatographic device for the separation of DNA fragments, proteins and peptides and other biological macromolecules to provide a fast and effective way. High-performance electrophoresis chromatography is the integration of gel electrophoresis resolution and fast liquid chromatography. When the sample is eluted from the gel, the continuous eluent flows with the separated components and passes a continuous detector. , Display the result and print the record. High-performance electrophoresis chromatography not only has the inherent high resolution and biocompatibility advantages of gel electrophoresis, but also facilitates continuous elution of samples.
First, the paper electrophoresis
1. The device includes electrophoresis chamber and DC power supply. The commonly used horizontal electrophoresis chamber device is shown in the figure, including two electrophoresis tanks A and one cover glass (or corresponding material) cover B; the electrophoresis tanks on both sides are divided into two parts by organic glass (or corresponding material) plate C; The outer grid is equipped with a platinum electrode (diameter 0.5-0.8cm) D; Rieger is an organic glass electrophoresis tank F which can put filter paper E. This rack can be taken out from the tank; the platinum electrode D inside the electrophoresis tank A on both sides is isolated The wire is connected to the external electrophoresis power supply through the slot wall. The power supply is a direct current power supply with a voltage regulator. Normal pressure electrophoresis is generally 100 to 500 V, and high voltage electrophoresis is generally 500 to 10 000 V.
2. Operation method
(1) Electrophoresis buffer citrate buffer (pH 3.0) 39.04 g of citric acid (C6H8O7·H2O) and 4.12 g of sodium decanoate (C6H5Na3O7·2H2O) were added, and 4,000 ml of water was added to dissolve the solution.
(2) filter paper filter paper filter set 1mol / L formic acid solution soak overnight, remove the next day, rinse with water until the pH of the lotion is not less than 4, set 60 °C oven drying, spare. According to the need to cut the growth of 27cm, width 18cm filter paper, or according to the size of the electrophoresis chamber cut, and the distance from the end of the end of the 5 ~ 8cm draw a starting line, every 2.5 ~ 3cm to make a mark for sampling purposes.
(3) There are wet spot method and dry spot method.
The wet spot method is to immerse the cut filter paper in the citrate buffer (pH 3.0), wet it, remove it, and blot excess buffer with filter paper to set the electrophoresis tank frame so that the starting line is close to the negative. Extremely, immerse the two ends of the filter paper in the buffer, and then use a microsyringe to precisely add the test solution, 10 μl per point, for a total of 3 points, and leave 2 blank positions.
The dry point method is to spot the test solution on the filter paper, blow dry, and then point again and again, until the point after the specified amount of the test solution, and then use a sprayer to spray the wet filter paper, the final spray wet spot, This law applies to dilute test solution.
(4) electrophoresis in electrophoresis tank by adding appropriate amount of electrophoresis buffer, immersed in platinum electrode, turn on the electrophoresis regulator power supply block, adjust the voltage gradient of 18 ~ 20V/cm, electrophoresis about 1 hour and 45 minutes, remove, blow dry immediately, Under UV light (254nm) view, use a pencil to draw purple spots.
(5) Determination of the content of the test sample and cut off the blank filter paper and the spot area is similar to the filter paper, cut into thin strips, respectively, placed in the test tube, each precision added 0.01mol/L hydrochloric acid solution 5ml, shake, place for 1 hour, with 3 The number of melting glass funnels is filtered. The supernatant can also be decanted by natural sedimentation or centrifugation. The absorbance is measured according to the provisions of each drug and the content is calculated according to the absorption coefficient.
Second, cellulose acetate membrane electrophoresis
1. Instrument equipment electrophoresis room and DC power with the paper electrophoresis.
2. Reagents
(1) Barbital Buffer (pH 8.6)
2.76 g of barbiturate and 15.45 g of pentobarbital sodium were dissolved and dissolved in water to make 1000 ml.
(2) Amino black dyeing solution 0.5 g of amino black 10B was dissolved in a mixture of 50 ml of methanol, 10 ml of glacial acetic acid, and 40 ml of water.
(3) Rinse the liquid with ethanol 45ml, glacial acetic acid 5ml and water 50ml, and mix.
(4) 25 ml of glacial acetic acid in a transparent solution, and 75 ml of anhydrous ethanol, and mix.
3. Operation method
(1) Cellulose Acetate Film Take Cellulose Acetate Film and Cut into 2cm×8cm Film Strips. Put the matte face down. Dip in a Barbital buffer solution (pH 8.6) until it is completely saturated. Remove and place it in the filter paper. After gently removing the excess buffer, the matte side of the film was placed on the electrophoresis tray and immersed in the buffer solution (pH 8.6) via a filter paper bridge.
(2) Spotting and electrophoresis are performed on the membrane strip at a distance of 2 cm from the negative electrode end, and the stripe-like protein solution with a content of about 5% is added to 2 to 3 μl of the sample solution and electrophoresed at a potential gradient of 10 to 12 V/cm. The electrophoresis zone distance is preferably 4 to 5 cm.
(3) After the dyeing and electrophoresis are completed, remove the membrane strip and immerse it in the amino black staining solution. After 2 to 3 minutes, rinse it several times with the rinsing solution until the background color is removed.
(4) Transparently immerse the washed and completely dried film in the transparent liquid for 10 to 15 minutes, remove the plate on a clean glass plate, and dry it to form a transparent film, which can be measured and made on a spectrophotometer. Specimens are kept for a long time.
(5) Determination of Cellulose Acetate Electrophoresis without Electrophoresis The electrophoretic patterns of cellulose acetate membranes that are not transparently treated can be determined according to the method specified under each drug. Generally, elution or scanning is used to determine the relative percentage of each protein component. In the elution method, the washed strips were blotted with filter paper, and the electrophoresis bands of the electrophoresis patterns of the test solutions were cut out, immersed in 1.6% sodium hydroxide solution, shaken several times, and eluted completely. The absorbance was measured at a certain wavelength. At the same time, the corresponding protein-free regions of the strips of the test article were cut and compared with the method of manipulation. The sum of the absorption values ​​was first calculated and then the percentage of each protein component was calculated. Scanning method The dried cellulose acetate film was automatically plotted on the recorder by reflection (non-transparent film) or transmissive (transparent film) chromatogram scanners with a chromatography scanner. The abscissa was the length of the film strip. The ordinate is absorbance and the percentage content of each protein component is calculated.
You can also use the computer to process the integral calculation.
1. Instrument equipment electrophoresis room and DC power with the paper electrophoresis.
2. Reagents
(1) Acetate-Lithium Salt Buffer (pH 3.0)
Take glacial acetic acid 50ml, add 800ml water, mix, adjust pH to 3.0 with lithium hydroxide, add water to
1000ml.
(2) Toluidine Blue Solution Take toluidine blue 0.1g and add 100ml of water to dissolve.
3. Operation method
(1) Take glue to take about 0.2g of agarose, add 10ml of water, set in water bath to swell completely, warm 10ml of hot acetic acid-lithium salt buffer solution (pH3.0), mix, and apply the glue liquid while hot. On a glass plate of suitable size (2.5cm x 7.5cm or 4cm x 9cm), the thickness is about 3mm, and it is left to stand until the gel forms a uniform thin layer without bubbles, that is, it is obtained. (2) The preparation of the standard solution and the test solution is made according to the provisions of each drug.
(3) Spotting and Electrophoresis The acetic acid-lithium salt buffer (pH 3.0) was added to the electrophoresis tank, and the gel plate was placed on the electrophoresis tray and immersed in the buffer via the filter paper bridge. 1 μl was spotted on the negative side of the gel plate, and the power was turned on immediately. Under the conditions of a voltage gradient of about 30 V/cm and a current intensity of 1 to 2 mA/cm, the electrophoresis was performed for about 20 minutes, and the power was turned off.
(4) Staining and decolorization Remove the gel plate and stain it with toluidine blue solution. Wash the excess stain with water until the background is colorless.
Fourth, polyacrylamide gel electrophoresis
1. The instrument device is usually composed of a steady flow electrophoresis device and a disc or plate electrophoresis tank. The electrophoresis chamber has two slots, upper and lower, and each slot has a fixed platinum electrode. The platinum electrode is connected to the electrophoresis device by an isolated wire.
2. Reagents
(1) Solution A
Take 36.6 g of trimethylolaminomethane, 0.23 ml of tetramethylethylenediamine, add 48 ml of a 0.1 mol/L hydrochloric acid solution, add water, dissolve and dilute to 100 ml, store in a brown bottle, and store in a refrigerator.
(2) Solution B
Take 30.0g of acrylamide and 0.74g of methylenebisacrylamide, add water to dissolve and dilute it to 100ml, filter it, place it in a brown bottle, and keep in a refrigerator.
(3) Electrode buffer (pH 8.3)
Take trishydroxymethyl aminomethane 6g, glycine 28.8g, dissolved in water and diluted to 1000ml, stored in the refrigerator, diluted 10 times before use.
(4) The bromophenol blue indicator solution to take bromophenol blue 0.1g, plus 0.05mol/L sodium hydroxide solution 3.0ml and 90% ethanol 5ml, slightly heat to dissolve, add 20% ethanol made 250ml.
(5) Staining solution: Take 0.25% (W/V) Coomassie Brilliant Blue G<[250]> solution 2.5 ml, and add 12.5% ​​(w/v) trichloroacetic acid solution to 10 ml.
(6) Dilute staining solution Take 2 ml of the above staining solution and add 12.5% ​​(w/v) trichloroacetic acid solution to 10 ml.
(7) Decolorizing liquid
(2) The preparation of the standard solution and the test solution follow the regulations for each drug.
(3) Electrophoresis Place the prepared gel glass tube into the electrophoresis tank of the disc, and add 50 to 100 μl of each sample or standard solution for each tube. Add glycerine or 40% sucrose solution to prevent 1-2 or 1 to 2 drops of the solution. And 0.04% bromophenol blue indicating solution 1 drop, but also directly in the upper tank buffer plus 0.04% bromophenol blue indicator solution a few drops, the top of the glass tube filled with electrode buffer, the upper end of the negative electrode, the lower end of the positive electrode. Adjust the initial current so that each tube is 1mA, and after a few minutes, increase the current so that each tube is 2 ~ 3mA, when the bromophenol blue indicator liquid is moved to 1cm away from the bottom of the glass tube, turn off the power.
(4) Dyeing and decolorization When the electrophoresis is completed, a syringe filled with long needles and filled with water is used to press the water along the wall of the hose from the bottom of the hose, the strip slides out from the tube, and the strip is immersed in a thin dyeing solution overnight or The dyeing solution is soaked for 10-30 minutes, rinsed with water, and then decolorized with a decolorizing solution until the protein-free gel-containing background is clear.
(5) Judgment of the result The tape is observed under the lamp and judged according to the position and color shade of the ribbon for the sample and the standard.
Relative Mobility The relative electrophoretic mobility (R''<[m]>) of the test sample and the standard can be compared when the electrophoresis zone is present. The calculation formula is as follows: The distance relative mobility (R''<[m]>)=- from the injecting end to the test article or the standard product area will be clearly cleared by the scanning distance from the injecting end to the bromophenol blue region. The strips were scanned and integrated in a dual-wavelength thin-layer scanner or gel electrophoresis scanner and the percentages were calculated from the peak areas of the components. 7% acetic acid solution.
3. Operation method
(1) Take glue to take solution A2ml, solution B5.4ml, add urea 2.9g to dissolve, add water 4ml, mix well, pump air to bubble in solution, add 0.5% ammonium persulfate solution 2ml, mix to make glue Immediately add liquid to the small glass tube (10 x 0.5 cm) with a rubber stopper at the bottom along the tube wall with a syringe or a fine dropper equipped with a long needle so that the adhesive layer height reaches 6 to 7 cm, then slowly A small amount of water was added dropwise to cover the plastic surface. The bubbles at the bottom of the tube must be driven off and allowed to stand for about 30 minutes. Polymerization was completed when an obvious interface appeared, and the water layer was sucked off.
Five, SDS polyacrylamide gel electrophoresis
The SDS polyacrylamide gel electrophoresis was used to determine the molecular weight of the protein, which is based on the fact that most of the proteins can be combined with the cationic surfactant sodium dodecyl sulfate (SDS) in a weight ratio to form a complex, so that the negative charge of the protein molecules is far away. Exceeding the negative charge of the natural protein molecule eliminates the charge effect of different protein molecules, so that the relative mobility of the protein molecule (R''<[m]>) depends entirely on the molecular weight and therefore can be determined from the known molecular weight standards. The molecular weight of the test sample is determined from the standard curve of the logarithm of the protein and the relative mobility.
1. Instrumentation Apparatus Polyacrylamide gel electrophoresis, unless otherwise specified.
2. Reagents
(1) Acrylamide solution Weigh 22.2 g of acrylamide and 0.6 g of bisacrylamide, dissolve in 100 ml of water and store in a brown bottle at low temperature.
(2) Gel Buffer Weigh 8.82g of sodium dihydrogen phosphate (NaH2PO4 · 2H2O), 51.55g of Na2HPO4 · 12H2O, and sodium dodecylsulfate 2.0g, and add water to 1000ml (if precipitated.) , can be heated to 37 °C to dissolve).
(3) Electrophoresis Buffer Diluted the gel buffer by 1 time.
(4) Staining solution Weigh 25 mg of Coomassie Brilliant Blue R<250> and dissolve in 100 ml of a mixture of 57% ethanol and 9.2% acetic acid.
(5) Decolorization solution: Take 75 ml of anhydrous ethanol, add 50 ml of glacial acetic acid, and dilute with water to 1000 ml.
3. The method of operation Except the following provisions, all other polyacrylamide gel electrophoresis.
(1) Glue is prepared from acrylamide solution-gel buffer-water-1.6% ammonium persulfate solution-tetramethylethylenediamine (need to be cooled) (10.1:15.0:3.4:1.5:0.045).
(2) Preparation of standard protein solution and test solution. Take standard protein, add water to make 2~5mg solution per 1ml, and hydrolyze solution (take urea 21.6g and sodium dodecyl sulfate 0.04g, dissolve 40 ml water) 1:3 mix, set in the refrigerator overnight. The test sample was prepared as described above.
(3) The electrophoresis adjusts the current to 8 mA per tube.
4. Calculation of Relative Mobility and Molecular Weight The electrophoretic decolorized zone was measured with a caliper or with a scanning positioning method to measure the distance the dye moved, the length of the strip before dyeing, the distance of protein movement and the length of the strip after decoloration. The relative mobility is calculated according to the following equation: Length of protein movement Distance of the strip before dyeing Relative mobility (R''<[m]>) =───×─── Distance of the front edge of the dye after the decolorization of the strip Using R''<[m]> as the abscissa, the logarithm of the known molecular weight standard protein is plotted on the ordinate, plotted on semi-logarithmic paper, and the molecular weight of the test sample is determined from the standard curve.