Electrophoresis electrophoresis refers to the action of charged electric test materials (proteins, nucleotides, etc.) in an inert support medium (such as paper, cellulose acetate, agarose gel, polyacrylamide gel, etc.). Next, the method of moving to the corresponding electrode direction at respective speeds to separate the components into narrow zones, recording the electrophoresis zone map or calculating the content (%) by a suitable detection method. Each electrophoresis method was operated as follows except as otherwise specified. The first method of paper electrophoresis 1. The instrument device comprises an electrophoresis chamber and a DC power supply. The commonly used horizontal electrophoresis chamber device comprises two electrophoresis tanks A and a sealable glass (or corresponding material) cover B; the electrophoresis tanks on both sides are divided into two parts by a plexiglass (or corresponding material) plate C; The outer grid is equipped with a platinum electrode (diameter 0.5-0.8 cm) D; the Rigg is a plexiglass electrophoresis trough F that can be placed on the filter paper E, and the rack can be taken out from the trough; the platinum electrode D in the electrophoresis tank A on both sides is isolated The wire is connected to the external electrophoresis power supply through the wall of the slot. The power supply is a DC power supply with a voltage regulator. The atmospheric pressure electrophoresis is generally between 100 and 500 V, and the high voltage electrophoresis is generally between 500 and 10 000 V. 2. Method of operation (1) Electrophoresis buffer citrate buffer (pH 3.0). 39.04 g of citric acid (C6H8O7?H2O) and 4.12 g of sodium citrate (C6H5Na3O7?2H2O) were added, and 4000 ml of water was added to dissolve. (2) The filter paper is taken from the chromatographic filter paper and immersed in a 1 mol/l formic acid solution overnight. The next day, it is taken out, rinsed with water until the pH of the washing liquid is not lower than 4, and dried in an oven at 60 ° C for use. It can be cut into 27cm and 18cm wide filter paper as needed, or cut according to the size of the electrophoresis chamber, and draw a starting line at 5~8cm from the end of the length direction, and make a mark every 2.5~3cm. (3) There are wet point method and dry point method. The wet point method is to immerse the cut filter paper in citrate buffer (pH 3.0), wet it, take it out, use the filter paper to blot out the excess buffer, and place it on the electrophoresis rack to make the starting line close to the yin. Extremely, immerse both ends of the filter paper in the buffer solution, then carefully add the test solution with a micro-injector, 10 μl per point, a total of 3 points, and leave 2 blank positions. The dry point method is to place the test solution on the filter paper, blow it dry, and then repeat it several times until the specified amount of the test solution is finished, then spray the filter paper with a sprayer, and finally spray the spot at the spot. This method applies to dilute test solutions. (4) Electrophoresis in the electrophoresis tank, add appropriate amount of electrophoresis buffer, immerse the platinum electrode, turn on the electrophoresis regulator power supply file, adjust the voltage gradient to 18 ~ 20V / cm, electrophoresis for about 1 hour and 45 minutes, take out, immediately blow dry, Place the UV lamp (254 nm) for inspection and use a pencil to mark the purple spot. (5) Determination of content Cut out the test strips and the blank filter paper with the area of ​​the spot position, cut into thin strips, place them in separate tubes, add 5ml of 0.01mol/l hydrochloric acid solution precisely, shake well, place for 1 hour, use 3 The fused glass funnel is filtered, and the supernatant can also be decanted by natural sedimentation or centrifugation. The absorbance is determined according to the regulations under each drug, and the content is calculated according to the absorption coefficient. The second method cellulose acetate film electrophoresis method 1. The instrument device electrophoresis chamber and DC power source are electrophoresed with paper. 2. Reagents (1) Barbiturate buffer (pH 8.6) Take 2.76 g of barbital and 15.45 g of barbital sodium, and dissolve in water to make 1000 ml. (2) Amino black staining solution 0.5 g of amino black 10B was taken and dissolved in a mixture of 50 ml of methanol, 10 ml of glacial acetic acid and 40 ml of water. (3) The rinse solution is taken with 45 ml of ethanol, 5 ml of glacial acetic acid and 50 ml of water, and mixed. (4) Take 25 ml of glacial acetic acid in clear liquid, add 75 ml of absolute ethanol, and mix. 3. Operation method (1) Cellulose acetate film Take cellulose acetate film, cut into 2cm × 8cm film strip, glazed face down, immersed in barbiturate buffer (pH8.6), to be completely saturated, take out After being sandwiched in the filter paper, the excess buffer was gently sucked off, and the film strip was placed with the matte side facing up, placed on the electrophoresis trough, and immersed in the barbital buffer (pH 8.6) through the filter paper bridge. (2) Spotting and electrophoresis on the strip 2 cm away from the negative end, and stripping 2 to 3 μl of the test solution with a protein content of about 5%, and electrophoresing at a potential gradient of 10 to 12 V/cm. The distance between the electrophoresis zones is preferably 4 to 5 cm. (3) After dyeing and electrophoresis, the strip is removed and immersed in the amino black staining solution. After 2 to 3 minutes, the strip is rinsed several times until the background color is removed. (4) Transparently immerse the cleaned and completely dried film strip in a transparent liquid for 10 to 15 minutes, remove it and lay it on a clean glass plate, and dry it to form a transparent film, which can be measured and measured on a spectrophotometer. Specimens are kept for a long time. (5) Determination of content The electrophoresis pattern of the cellulose acetate film which has not been transparently treated can be determined according to the method specified in each drug item, and the relative content (1%) of each protein component is generally determined by an elution method or a scanning method. The elution method is to dry the washed membrane strip with a filter paper, and the electrophoresis zone of each electrophoresis pattern of the test solution is cut out, immersed in a 1.6% sodium hydroxide solution, shaken several times, until the elution is complete. The absorbance is measured at a certain wavelength. At the same time, the protein-free part corresponding to the test strip is cut and compared with the same method. Calculate the sum of the absorption values ​​first, and then calculate the ratio of each protein component (1%). The third method SDS-polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis separation of proteins based on the principle that most proteins can be combined with the cationic surfactant sodium dodecyl sulfate (SDS) by weight ratio The complex is such that the negative charge carried by the protein molecule far exceeds the net charge of the natural protein molecule, eliminating the charge effect of different protein molecules and separating the proteins by molecular size. 1. The instrument is equipped with a constant voltage or constant current power supply, a vertical plate or a disk electrophoresis tank and a glue making mold. 2. Reagents (1) 30% acrylamide solution 60 g of acrylamide and 1.6 g of methylene bis acrylamide were added, water was added to 200 ml, filtered through a filter paper, and stored in the dark. (2) Separating the gel buffer, taking 36.3 g of trishydroxymethylaminomethane, adding 70 ml of water, adjusting the pH to 8.8 with hydrochloric acid, and adding water to 100 ml. (3) Concentrated gel buffer solution: 6.0 g of trishydroxymethylaminomethane, 70 ml of water, adjusted to pH 6.8 with hydrochloric acid, and added to 100 ml of water. (4) Electrophoresis buffer solution: 6.0 g of trishydroxymethylaminomethane, 28.8 g of glycine, 1.0 g of sodium lauryl sulfate, and water was added to 1000 ml. 3. Operation method (1) 30% acrylamide solution for gel making - separation gel buffer -20% sodium lauryl sulfate solution - 10% ammonium persulfate solution (freshly prepared) - tetramethylethylenediamine-water (5.0:1.5:0.08:0.1:0.01:5.3) The separation glue was prepared and poured into a mold to a certain height (the remaining volume was reserved for the preparation of concentrated glue), capped with water, and the polymerization was completed, and the water layer was decanted. Re-use 30% acrylamide solution - concentrated gel buffer - 20% sodium lauryl sulfate solution - 10% ammonium persulfate solution - tetramethylethylene diamine - water (0.8: 1.3: 0.025: 0.05: 0.005: 2.4 The concentrated glue is prepared, poured on the separation gel, and inserted into the sample comb (for example, disk electrophoresis, capped with water), and after the concentrated glue is polymerized, the sample comb or water is carefully removed. (2) Preparation of the reference substance and the test solution according to the provisions of each drug. (3) Electrophoresis vertical plate electrophoresis: constant pressure electrophoresis, the initial voltage is 80V, and it is adjusted to 150-200V when entering the separation gel. When the bromophenol blue migrates to the bottom of the gel, the electrophoresis is stopped. Disk electrophoresis: Adjust the current to 8 mA per tube. 4. Fixation and staining (1) Coomassie brilliant blue staining 1 reagent a. Fix the solution to weigh 5 g of trichloroacetic acid, add 200 ml of water to dissolve, add 200 ml of methanol, and add water to 500 ml. b. The staining solution was weighed with Coomassie Brilliant Blue R 0.5g, dissolved in 200 ml of water, and then added with 200 ml of methanol and 50 ml of glacial acetic acid, and then added with water to 500 ml. c. Decoloring solution Take 400 ml of methanol, 100 ml of glacial acetic acid, add water to 1000 ml, and mix well. d. Take 75 ml of glacial acetic acid in the preservation solution, add water to 1000 ml, and shake well. 2 Fixation and staining electrophoresis, remove the film (strip), set the fixative solution for 30 minutes, remove the film (strip), place the stain solution for 1-2 hours, decolorize with decolorization solution until the gel background is transparent and store in the preservation solution. . (2) Silver staining 1 reagent a. Silver nitrate solution 0.8g of silver nitrate, add water to 4.0ml, this solution is added dropwise to a mixture of 0.1ml / L sodium hydroxide solution 20ml and 25% ammonia solution 1.5ml, Shake well and dilute to 100 ml with water. b. Fix the solution with 50 ml of methanol and 54 μl of 37% formaldehyde solution, and add water to 100 ml. c. The coloring solution was taken as 1 ml of a 1% citric acid solution, 270 μl of a 37% formaldehyde solution, and water was added to 500 ml. d. Stop the solution and take 100 ml of glacial acetic acid and add water to 1000 ml. 2 Fix and dye the film in the fixing solution for at least 2 hours, discard the fixing solution, and dip it with water for at least 1 hour; place the film in 1% glutaraldehyde solution for 15 minutes, then wash twice with water for 15 minutes each time; After 15 minutes in the silver nitrate solution, it was washed 3 times with water for 15 minutes each time; the film was placed in the coloring solution, and the strips were exposed to the post-stop solution. 5. Calculate the bromophenol blue indicator and protein migration distance by caliper or by scanning and localization method (for the disc electrophoresis, the length of the strip before and after dyeing should be measured. The thickness of the vertical plate electrophoresis film is less than 1mm, and the film length before and after dyeing is basically not change). Calculate the relative mobility by the following formula: Protein migration distance Relative strip mobility before decolorization (R) = Length of strip after decolorization / Migration distance of bromophenol blue indicator (1) Molecular weight with R as abscissa, molecular weight of standard protein The logarithm is the ordinate, linear regression is performed, and the molecular weight of the test sample is obtained from the standard curve. (2) Purity film (strip) is placed, and a thin layer scanner is placed, and the peak area is calculated by the normalization method. (3) The result judges that the main component mobility of the test product should be consistent with the reference product mobility.
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