CN110554081A - Isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein - Google Patents
Isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein Download PDFInfo
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Abstract
The invention discloses an isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein, which comprises the following steps: s1, preparing materials; various materials used in the experiment are properly prepared to ensure that the experiment is smoothly carried out; s2, preparing a reagent; reagents required by the experiment, namely a sample to be detected and a reference substance, need to be prepared in time for the experiment; s3, preparing an instrument; the experimental instrument is prepared after being cleaned in advance; s4, preparing a reagent; the reagent which can not be directly used in the experiment is manually prepared for use, so that the smooth operation of the experiment is ensured; s5, a detection method; mainly takes electrophoresis as a method to test the test; s6, analyzing and judging results; the result is judged by comparing the analysis of the reference substance; the invention covers the preparation of materials, reagents and instruments, the reasonable preparation of the reagents, a scientific inspection method and rigorous analysis and judgment, and the detection of isoelectric points of the recombinant protein by isoelectric focusing electrophoresis is carried out, so that the result is scientific and reasonable and has convincing effect.
Description
Technical Field
the invention belongs to the technical field of isoelectric focusing electrophoresis isoelectric point detection of recombinant proteins, and particularly relates to an isoelectric focusing electrophoresis isoelectric point detection method of recombinant proteins.
Background
isoelectric focusing (IEF) is a new technology that has emerged in the mid sixties. In recent years, the isoelectric focusing technology has been developed and rapidly developed into a mature modern biochemical experimental technology. The method has the advantages of high resolution, good repeatability, large sample capacity and simple and rapid operation, and is widely applied to biochemical, molecular biology and clinical medicine research.
therefore, an isoelectric focusing electrophoresis method of S Mm BS-4 is developed on the basis of the 2 nd method of the isoelectric focusing electrophoresis method of the three parts of Chinese pharmacopoeia of 2015 edition, and in order to improve the separation degree and the repeatability of the method, the original isoelectric focusing electrophoresis method is optimized and verified at present, and a more stable and reliable analysis method is planned to be established and used for quality control of isoelectric points of later-stage recombinant human erythropoietin (Fc) fusion protein stock solution (rhEPO-Fc).
Although the isoelectric point detection of the protein is carried out by establishing an isoelectric focusing electrophoresis analysis method of polyacrylamide and the like, the resolution of the method is still required to be further improved, the isoelectric focusing test of 'recombinant human erythropoietin injection (CHO cell)' recorded in the edition of Chinese pharmacopoeia 2015 contains urea, so that the isoelectric focusing electrophoresis is carried out by adding urea and not adding urea in the isoelectric focusing formula in the research of a recombinant human erythropoietin quality control system, and as a result, the resolution of each band of the added urea is obviously improved.
Disclosure of Invention
the invention aims to: in order to solve the problem of isoelectric point detection of isoelectric focusing electrophoresis of recombinant protein, a method for detecting isoelectric point of isoelectric focusing electrophoresis of recombinant protein is provided.
the technical scheme adopted by the invention is as follows:
an isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein comprises the following steps:
s1, material preparation: various materials used in the experiment are properly prepared to ensure that the experiment is smoothly carried out;
s2, preparation of reagents: reagents required by the experiment, namely a sample to be detected and a reference substance, need to be prepared in time for the experiment;
s3, preparing an instrument: the experimental instrument is prepared after being cleaned in advance;
s4, preparation of reagents: the reagent which can not be directly used in the experiment is manually prepared for use, so that the smooth operation of the experiment is ensured;
s5, a detection method: mainly takes electrophoresis as a method to test the test;
s6, analyzing and judging the result: the result is judged by comparing the analysis of the reference substance;
the preparation method of the S1 material comprises the following specific steps:
s101, preparing a polypropylene film: PAG fim (polypropylene film, GE healthcare);
S102, manually cutting the electrode strip: the electrode strip is cut manually and can be cut according to the requirement;
s103, manually cutting sample paper: the sample loading paper needs to be cut manually to ensure the smooth sample loading;
s104, manually cutting a dry adhesive film: the dry film was cut manually for later use.
wherein, the preparation of the S2 reagent comprises the following specific steps:
s201, preparing a sample to be detected and a reference substance: preparing a sample to be detected and a working reference substance for an experiment;
s202, preparing various experimental reagents: preparing acrylamide (electrophoresis grade); methylene bisacrylamide (electrophoresis grade); ammonia persulfate; trichloroacetic acid; ammonium sulfate; phosphoric acid (85%); sodium hydroxide; methanol; absolute ethyl alcohol; coomassie brilliant blue G250; glycerol; glacial acetic acid; a hydrophobic silane; TEMED; ampholytes (pH3.0-10.0, pH4.0-6.5); marker (3-10); ultrapure water, and the like.
The S3 instrument is prepared, and the specific steps are as follows:
s301, preparing an isoelectric focusing device: isoelectric focusing devices (Multiphor II multifunctional electrophoresis systems, GE; small cooling water circulation devices, EYELA);
S302, preparing a laser scanning densitometer: Bio-Rad laser scanning densitometer (GS 800);
s303, preparing a horizontal oscillator;
The S4 reagent is prepared by the following specific steps:
s401, preparing a 30% acrylamide solution: weighing 58.2g of acrylamide and 1.8g of methylene bisacrylamide, dissolving with 150ml of pure water, then fixing the volume to 200ml, filtering by a 0.22 mu m membrane, and storing in the dark at 5 +/-3 ℃;
s402, preparing a stationary liquid: trichloroacetic acid mother liquor: weighing 100g of trichloroacetic acid, adding 100mL of water, mixing, and uniformly mixing before use; 20% trichloroacetic acid: mixing 20mL of mother liquor with 80mL of water (prepared as before use);
S403, preparing a dyeing solution: dyeing stock solution: weighing 125g of ammonium sulfate, and dissolving with 400ml of pure water to obtain solution 1; weighing 1.0G of Coomassie brilliant blue G250, adding 20ml of pure water, stirring, adding 20G of phosphoric acid, mixing with the solution 1 after dissolving, metering the volume to 1000ml, and fully shaking up before use; working dyeing liquid: taking 60ml of dyeing stock solution, mixing with 30ml of methanol uniformly, and carrying out new preparation for use;
s404, preparing 10% APS: 0.1g of sub-packaged ammonium persulfate is taken and dissolved in 1ml of ultrapure water, and the ammonium persulfate is prepared as it is (or stored at 5 +/-3 ℃ for 1 week for use);
S405, preparing an electrode solution: anode solution: measuring 57.6mL of phosphoric acid in 500mL of water, metering to 1000mL of phosphoric acid solution at 0.5mol/L, filtering with a 0.22 mu m membrane, and storing at normal temperature; and (3) negative electrode solution: weighing 8g of sodium hydroxide in 0.2mol/L sodium hydroxide solution, dissolving in 500mL of water, diluting to 1000mL, filtering with 0.22 μm membrane, and storing at normal temperature;
S406, preparing 80% glycerol: measuring 160ml of glycerol, adding pure water to dilute the glycerol to 200ml, filtering the glycerol by using a 0.45-micron membrane, and storing the glycerol at normal temperature;
s407, preparing a decoloring solution: preparing 250ml of 95% ethanol, 80ml of glacial acetic acid and 670ml of ultrapure water;
S408, preparing a dry glue dehydration solution: 200ml of absolute ethyl alcohol, 50ml of glacial acetic acid, 50ml of glycerol and 100ml of ultrapure water are uniformly mixed and the volume is up to 500 ml.
the S5 test method comprises the following specific steps:
s501, assembling a glue making frame: putting a clean glue-making glass plate on clean wiping cloth, uniformly coating 0.5mL of hydrophobic silane on the front surface of a rubber strip glass plate with the mu m, and airing at room temperature; adding 0.5ml of water to one surface of a plane glass plate for wetting, taking a glue making film, sticking a glass plate on a hydrophobic surface, enabling the hydrophobic surface to be tightly attached to the wetted glass plate by using a manual roller press and removing bubbles completely, and cleaning redundant water by using absorbent paper; two glass plates are symmetrically fixed by using a glue-making clamp at the left side and the right side respectively, and the lower side of the glue-making clamp is two glass plates (pre-cooled at 5 +/-3 ℃ when the room temperature is too high);
s502, preparing and filling a glue solution: urea mother liquor: weighing 10.8g of urea, directly adding 17.5mL of ultrapure water to dissolve the urea to 24.6mL, and filtering by a 0.22 mu m membrane for later use; preparing glue and filling: 3.3ml of 30% polyacrylamide solution, 12.3ml of urea mother liquor, 1.2ml of 80% glycerol, 0.275ml of pH4.0-6.5 electrolyte, 0.825ml of pH3.0-10.0 electrolyte and 100 mul of 10% APS, uniformly mixing, adding 20 mul of TEMED, uniformly mixing, sucking the mixed solution by using a 5ml liquid-transferring gun, filling the mixed solution into gaps of assembled glue-making molds, carefully avoiding bubbles (if bubbles are generated, slightly beating a glass plate by hands, discharging bubbles), standing at room temperature for 1.5h, completely solidifying (when the temperature is too low, standing at 25 ℃ for 2.5 h), immediately using or wrapping by using a preservative film, and storing at 5 +/-3 ℃ for 2-3 days;
s503, pre-electrophoresis: opening a switch of the small cooling water circulation device until the temperature is cooled to 16 ℃, then placing 1mL of ultrapure water on an electrophoresis plate, taking glue, symmetrically sticking the glue on 3-13 parts of the electrophoresis plate, and removing bubbles; and (3) placing an electrode strip (shorter than the glue) at the transverse lines of 3 and 13, adding an appropriate amount (about 1 mL) of negative electrode solution in the negative electrode direction until the electrode strip is wetted, and adding an appropriate amount (about 1 mL) of positive electrode solution in the positive electrode direction until the electrode strip is wetted. Covering an electrophoresis tank cover, opening a switch of an electrophoresis power supply device to set 700V, 24mA and 8W, and pre-electrophoresis for 60 min;
s504, sample treatment: dissolving a control sample with 250ml of ultrapure water to obtain a control sample with the concentration of 2 mg/ml; diluting the sample to 2 mg/ml;
s505, spotting: placing a sample paper sheet (cut into a square of about 0.5X1.0cm by using a piece of lens wiping paper) on the gel at a position 6cm away from the cathode, and spotting a Marker; placing a sample loading paper sheet, a sample application sample and a working reference substance on the gel at a position 1cm away from the cathode, wherein the sample application amount is 20 mu g (the volume is 10 mu l);
s506, electrophoresis: loading and electrophoresis: setting 500V, 8mA, 8W, temperature 10-20 ℃ (general 16 ℃), and electrophoresis for 20 min. Removing the sample loading paper sheet after finishing; formal electrophoresis: setting 2200V, 14mA, 18W, 180min +2500V, 14mA, 20W, 10min, and 10-20 ℃ (generally 16 ℃);
s507, dyeing and decoloring: after electrophoresis, taking a film, enabling the rubber surface to face upwards, placing the film in a fixing solution, oscillating and fixing the film for 20min at room temperature and 10rpm, oscillating and cleaning the film for 2min with clear water after the electrophoresis is finished, then oscillating and dyeing the film in a dyeing solution for 1 h until bands are clearly shown (when the temperature in winter is too low, the dyeing time is properly prolonged until the bands are clearly shown), rinsing redundant dyeing solution with clear water when dyeing is stopped, placing the film in clear water, slightly wiping the film with a common paper towel to remove floating color (when wiping, keeping the film soaked in the water to keep moist so as not to wipe the rubber surface), and placing the film in a decoloring solution to remove a background;
S508, glue sweeping: GS800 scanning analysis isoelectric point;
S509, preparing dry glue: placing the film in a small amount of dry glue dewatering liquid, oscillating for 5min, placing on a glass plate, sticking a dry glue film, sealing the periphery with transparent glue, and naturally drying in a fume hood.
during preparation and filling of the S502 glue solution, if bubbles are generated, slightly knocking the glass plate by hands to discharge the bubbles; when the temperature is too low, the mixture is placed in an environment with 25 ℃ for solidification for 2.5 hours.
wherein, in the S503 pre-electrophoresis, 700V, 24mA and 8W are set; during electrophoresis, attention should be paid to the change of relevant parameters such as voltage, and when electrophoresis is finished, the voltage should reach the set upper limit voltage.
in the S506 electrophoresis, the sample loading electrophoresis is set to be 500V, 8mA and 8W; the general whole gel set for formal electrophoresis is 2200V, 14mA, 18W, 180min +2500V, 14mA, 20W, 10 min.
in the S507 dyeing and decoloring, when the temperature is too low in winter, the dyeing time is properly prolonged until the strips are clearly shown; the rubber sheet should be kept wet by soaking in water during wiping so as to avoid rubbing the rubber surface.
in the results of the analysis and determination of S6, each sample group must contain at least one control, and each lane should be vertical without diffusion and be well separated; the bands of the reference substance should be distributed within the range of pI 4.6-6.8, and the migration distance of the main components of the sample should be consistent with that of the reference substance.
in summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the invention provides a method for detecting isoelectric points of isoelectric focusing electrophoresis of recombinant proteins, which comprises the steps of preparing materials, reagents and instruments, reasonably preparing the reagents, scientifically detecting the reagents, strictly analyzing and judging, carrying out isoelectric focusing electrophoresis on the recombinant proteins, and having scientific, reasonable and convincing results.
2. The material of the invention is prepared by the following steps: preparing a polypropylene film; PAG fim (polypropylene film, GE healthcare); electrode strips, sample paper and dry adhesive films are cut manually to ensure that required materials are cut according to the requirements of the materials, and the experiment can be completed more effectively.
3. the invention further provides for the apparatus to be: preparing an isoelectric focusing device (Multiphor II multifunctional electrophoresis System, GE; Small Cooling Water circulation device, EYELA); preparing a laser scanning densitometer; Bio-Rad laser scanning densitometer (GS 800); and (5) preparing a horizontal oscillator, cleaning and checking the instrument in advance, and preparing for an experiment.
4. The invention further provides reagent preparation, wherein the preparation of a sample to be detected and a reference substance comprises the following steps: preparing a sample to be detected and a working reference substance for an experiment; preparing various experimental reagents; preparing acrylamide (electrophoresis grade); methylene bisacrylamide (electrophoresis grade); ammonia persulfate; trichloroacetic acid; ammonium sulfate; phosphoric acid (85%); sodium hydroxide; methanol; absolute ethyl alcohol; coomassie brilliant blue G250; glycerol; glacial acetic acid; a hydrophobic silane; TEMED; ampholytes (pH3.0-10.0, pH4.0-6.5); marker (3-10); ultrapure water and the like are prepared in order to ensure that the experiment can be smoothly carried out.
5. the invention further discloses a reagent for preparing the reagent, wherein a 30% acrylamide solution is prepared by the following steps: weighing 58.2g of acrylamide and 1.8g of methylene bisacrylamide, dissolving with 150ml of pure water, then fixing the volume to 200ml, filtering by a 0.22 mu m membrane, and storing in the dark at 5 +/-3 ℃; preparing a stationary liquid; trichloroacetic acid mother liquor: weighing 100g of trichloroacetic acid, adding 100mL of water, mixing, and uniformly mixing before use; 20% trichloroacetic acid: mixing 20mL of mother liquor with 80mL of water (prepared as before use); preparing a dyeing solution; dyeing stock solution: weighing 125g of ammonium sulfate, and dissolving with 400ml of pure water to obtain solution 1; weighing 1.0G of Coomassie brilliant blue G250, adding 20ml of pure water, stirring, adding 20G of phosphoric acid, mixing with the solution 1 after dissolving, metering the volume to 1000ml, and fully shaking up before use; working dyeing liquid: taking 60ml of dyeing stock solution, mixing with 30ml of methanol uniformly, and carrying out new preparation for use; preparing 10% APS; 0.1g of sub-packaged ammonium persulfate is taken and dissolved in 1ml of ultrapure water, and the ammonium persulfate is prepared as it is (or stored at 5 +/-3 ℃ for 1 week for use); preparing an electrode solution; anode solution: measuring 57.6mL of phosphoric acid in 500mL of water, metering to 1000mL of phosphoric acid solution at 0.5mol/L, filtering with a 0.22 mu m membrane, and storing at normal temperature; and (3) negative electrode solution: weighing 8g of sodium hydroxide in 0.2mol/L sodium hydroxide solution, dissolving in 500mL of water, diluting to 1000mL, filtering with 0.22 μm membrane, and storing at normal temperature; preparing 80% glycerol; measuring 160ml of glycerol, adding pure water to dilute the glycerol to 200ml, filtering the glycerol by using a 0.45-micron membrane, and storing the glycerol at normal temperature; preparing a decoloring solution; preparing 250ml of 95% ethanol, 80ml of glacial acetic acid and 670ml of ultrapure water; preparing a dry glue dehydrating solution; 200ml of absolute ethyl alcohol, 50ml of glacial acetic acid, 50ml of glycerol and 100ml of ultrapure water are prepared and mixed uniformly, the volume is determined to be 500ml, the reagent is prepared in strict proportion and quantity, and the reagent required by the experiment is prepared scientifically and reasonably.
6. The invention further discloses a detection method, which comprises the following steps of: putting a clean glue-making glass plate on clean wiping cloth, uniformly coating 0.5mL of hydrophobic silane on the front surface of a rubber strip glass plate with the mu m, and airing at room temperature; adding 0.5ml of water to one surface of a plane glass plate for wetting, taking a glue making film, sticking a glass plate on a hydrophobic surface, enabling the hydrophobic surface to be tightly attached to the wetted glass plate by using a manual roller press and removing bubbles completely, and cleaning redundant water by using absorbent paper; two glass plates are symmetrically fixed by using a glue-making clamp at the left side and the right side respectively, and the lower side of the glue-making clamp is two glass plates (pre-cooled at 5 +/-3 ℃ when the room temperature is too high); preparing and filling a glue solution: urea mother liquor: weighing 10.8g of urea, directly adding 17.5mL of ultrapure water to dissolve the urea to 24.6mL, and filtering by a 0.22 mu m membrane for later use; preparing glue and filling: 3.3ml of 30% polyacrylamide solution, 12.3ml of urea mother liquor, 1.2ml of 80% glycerol, 0.275ml of pH4.0-6.5 electrolyte, 0.825ml of pH3.0-10.0 electrolyte and 100 mul of 10% APS, uniformly mixing, adding 20 mul of TEMED, uniformly mixing, sucking the mixed solution by using a 5ml liquid-transferring gun, filling the mixed solution into gaps of assembled glue-making molds, carefully avoiding bubbles (if bubbles are generated, slightly beating a glass plate by hands, discharging bubbles), standing at room temperature for 1.5h, completely solidifying (when the temperature is too low, standing at 25 ℃ for 2.5 h), immediately using or wrapping by using a preservative film, and storing at 5 +/-3 ℃ for 2-3 days; pre-electrophoresis: opening a switch of the small cooling water circulation device until the temperature is cooled to 16 ℃, then placing 1mL of ultrapure water on an electrophoresis plate, taking glue, symmetrically sticking the glue on 3-13 parts of the electrophoresis plate, and removing bubbles; and (3) placing an electrode strip (shorter than the glue) at the transverse lines of 3 and 13, adding an appropriate amount (about 1 mL) of negative electrode solution in the negative electrode direction until the electrode strip is wetted, and adding an appropriate amount (about 1 mL) of positive electrode solution in the positive electrode direction until the electrode strip is wetted. Covering an electrophoresis tank cover, opening a switch of an electrophoresis power supply device to set 700V, 24mA and 8W, and pre-electrophoresis for 60 min; processing a sample; dissolving a control sample with 250ml of ultrapure water to obtain a control sample with the concentration of 2 mg/ml; diluting the sample to 2 mg/ml; sample application: placing a sample paper sheet (cut into a square of about 0.5X1.0cm by using a piece of lens wiping paper) on the gel at a position 6cm away from the cathode, and spotting a Marker; placing a sample loading paper sheet, a sample application sample and a working reference substance on the gel at a position 1cm away from the cathode, wherein the sample application amount is 20 mu g (the volume is 10 mu l); electrophoresis: loading and electrophoresis: setting 500V, 8mA, 8W, temperature 10-20 ℃ (general 16 ℃), and electrophoresis for 20 min. Removing the sample loading paper sheet after finishing; formal electrophoresis: setting 2200V, 14mA, 18W, 180min +2500V, 14mA, 20W and 10min), and setting the temperature to be 10-20 ℃ (generally 16 ℃); dyeing and decoloring: after electrophoresis, taking a film, enabling the rubber surface to face upwards, placing the film in a fixing solution, oscillating and fixing the film for 20min at room temperature and 10rpm, oscillating and cleaning the film for 2min with clear water after the electrophoresis is finished, then oscillating and dyeing the film in a dyeing solution for 1 h until bands are clearly shown (when the temperature in winter is too low, the dyeing time is properly prolonged until the bands are clearly shown), rinsing redundant dyeing solution with clear water when dyeing is stopped, placing the film in clear water, slightly wiping the film with a common paper towel to remove floating color (when wiping, keeping the film soaked in the water to keep moist so as not to wipe the rubber surface), and placing the film in a decoloring solution to remove a background; glue sweeping: GS800 scanning analysis isoelectric point; and (3) drying glue: placing the film in a small amount of dry glue dehydration solution, oscillating for 5min, placing on a glass plate, sticking a dry glue film, sealing the periphery with transparent glue, and naturally drying in a fume hood; the test is mainly carried out by electrophoresis so as to achieve the purpose of experiment.
7. according to the further analysis and judgment result, each sample group must contain at least one reference substance, and each lane should be vertical without diffusion and well separated; the bands of the reference substance are distributed within the range of pI 4.6-6.8, the migration distance of the main components of the sample is consistent with that of the reference substance, and the limiting conditions of the sample and the reference substance are favorable for better comparison.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a schematic diagram of a material preparation process according to the present invention;
FIG. 3 is a schematic diagram of a reagent preparation process according to the present invention;
FIG. 4 is a schematic diagram of the apparatus preparation process of the present invention;
FIG. 5 is a schematic diagram of a process for preparing reagents according to the present invention;
FIG. 6 is a schematic flow chart of the inspection method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
in the first embodiment, referring to fig. 1-2, a method for detecting isoelectric points of recombinant proteins by isoelectric focusing electrophoresis comprises the following steps:
s1, material preparation: various materials used in the experiment are properly prepared to ensure that the experiment is smoothly carried out;
S2, preparation of reagents: reagents required by the experiment, namely a sample to be detected and a reference substance, need to be prepared in time for the experiment;
s3, preparing an instrument: the experimental instrument is prepared after being cleaned in advance;
S4, preparation of reagents: the reagent which can not be directly used in the experiment is manually prepared for use, so that the smooth operation of the experiment is ensured;
s5, a detection method: mainly takes electrophoresis as a method to test the test;
s6, analyzing and judging the result: the result is judged by comparing the analysis of the reference substance;
the preparation method of the S1 material comprises the following specific steps:
s101, preparing a polypropylene film: PAG fim (polypropylene film, GE healthcare);
S102, manually cutting the electrode strip: the electrode strip is cut manually and can be cut according to the requirement;
s103, manually cutting sample paper: the sample loading paper needs to be cut manually to ensure the smooth sample loading;
s104, manually cutting a dry adhesive film: the dry film was cut manually for later use.
in a second embodiment, referring to fig. 1 to 3, a method for detecting isoelectric points of recombinant proteins by isoelectric focusing electrophoresis comprises the following steps:
s1, material preparation: various materials used in the experiment are properly prepared to ensure that the experiment is smoothly carried out;
s2, preparation of reagents: reagents required by the experiment, namely a sample to be detected and a reference substance, need to be prepared in time for the experiment;
s3, preparing an instrument: the experimental instrument is prepared after being cleaned in advance;
s4, preparation of reagents: the reagent which can not be directly used in the experiment is manually prepared for use, so that the smooth operation of the experiment is ensured;
s5, a detection method: mainly takes electrophoresis as a method to test the test;
s6, analyzing and judging the result: the result is judged by comparing the analysis of the reference substance;
the preparation method of the S1 material comprises the following specific steps:
S101, preparing a polypropylene film: PAG fim (polypropylene film, GE healthcare);
s102, manually cutting the electrode strip: the electrode strip is cut manually and can be cut according to the requirement;
S103, manually cutting sample paper: the sample loading paper needs to be cut manually to ensure the smooth sample loading;
S104, manually cutting a dry adhesive film: manually cutting the dry adhesive film for later use;
Wherein, the preparation of the S2 reagent comprises the following specific steps:
s201, preparing a sample to be detected and a reference substance: preparing a sample to be detected and a working reference substance for an experiment;
s202, preparing various experimental reagents: preparing acrylamide (electrophoresis grade); methylene bisacrylamide (electrophoresis grade); ammonia persulfate; trichloroacetic acid; ammonium sulfate; phosphoric acid (85%); sodium hydroxide; methanol; absolute ethyl alcohol; coomassie brilliant blue G250; glycerol; glacial acetic acid; a hydrophobic silane; TEMED; ampholytes (pH3.0-10.0, pH4.0-6.5); marker (3-10); ultrapure water.
in a third embodiment, referring to fig. 1 to 4, a method for detecting isoelectric points of recombinant proteins by isoelectric focusing electrophoresis comprises the following steps:
s1, material preparation: various materials used in the experiment are properly prepared to ensure that the experiment is smoothly carried out;
s2, preparation of reagents: reagents required by the experiment, namely a sample to be detected and a reference substance, need to be prepared in time for the experiment;
s3, preparing an instrument: the experimental instrument is prepared after being cleaned in advance;
s4, preparation of reagents: the reagent which can not be directly used in the experiment is manually prepared for use, so that the smooth operation of the experiment is ensured;
s5, a detection method: mainly takes electrophoresis as a method to test the test;
s6, analyzing and judging the result: the result is judged by comparing the analysis of the reference substance;
the preparation method of the S1 material comprises the following specific steps:
s101, preparing a polypropylene film: PAG fim (polypropylene film, GE healthcare);
s102, manually cutting the electrode strip: the electrode strip is cut manually and can be cut according to the requirement;
S103, manually cutting sample paper: the sample loading paper needs to be cut manually to ensure the smooth sample loading;
s104, manually cutting a dry adhesive film: manually cutting the dry adhesive film for later use;
Wherein, the preparation of the S2 reagent comprises the following specific steps:
s201, preparing a sample to be detected and a reference substance: preparing a sample to be detected and a working reference substance for an experiment;
s202, preparing various experimental reagents: preparing acrylamide (electrophoresis grade); methylene bisacrylamide (electrophoresis grade); ammonia persulfate; trichloroacetic acid; ammonium sulfate; phosphoric acid (85%); sodium hydroxide; methanol; absolute ethyl alcohol; coomassie brilliant blue G250; glycerol; glacial acetic acid; a hydrophobic silane; TEMED; ampholytes (pH3.0-10.0, pH4.0-6.5); marker (3-10); ultrapure water;
The preparation method of the S3 instrument comprises the following specific steps:
s301, preparing an isoelectric focusing device: isoelectric focusing devices (Multiphor II multifunctional electrophoresis systems, GE; small cooling water circulation devices, EYELA);
s302, preparing a laser scanning densitometer: Bio-Rad laser scanning densitometer (GS 800);
and S303, preparing a horizontal oscillator.
in a fourth embodiment, referring to fig. 1 to 5, a method for detecting isoelectric points of recombinant proteins by isoelectric focusing electrophoresis comprises the following steps:
s1, material preparation: various materials used in the experiment are properly prepared to ensure that the experiment is smoothly carried out;
s2, preparation of reagents: reagents required by the experiment, namely a sample to be detected and a reference substance, need to be prepared in time for the experiment;
s3, preparing an instrument: the experimental instrument is prepared after being cleaned in advance;
s4, preparation of reagents: the reagent which can not be directly used in the experiment is manually prepared for use, so that the smooth operation of the experiment is ensured;
S5, a detection method: mainly takes electrophoresis as a method to test the test;
S6, analyzing and judging the result: the result is judged by comparing the analysis of the reference substance;
the preparation method of the S1 material comprises the following specific steps:
s101, preparing a polypropylene film: PAG fim (polypropylene film, GE healthcare);
s102, manually cutting the electrode strip: the electrode strip is cut manually and can be cut according to the requirement;
s103, manually cutting sample paper: the sample loading paper needs to be cut manually to ensure the smooth sample loading;
s104, manually cutting a dry adhesive film: manually cutting the dry adhesive film for later use;
Wherein, the preparation of the S2 reagent comprises the following specific steps:
s201, preparing a sample to be detected and a reference substance: preparing a sample to be detected and a working reference substance for an experiment;
s202, preparing various experimental reagents: preparing acrylamide (electrophoresis grade); methylene bisacrylamide (electrophoresis grade); ammonia persulfate; trichloroacetic acid; ammonium sulfate; phosphoric acid (85%); sodium hydroxide; methanol; absolute ethyl alcohol; coomassie brilliant blue G250; glycerol; glacial acetic acid; a hydrophobic silane; TEMED; ampholytes (pH3.0-10.0, pH4.0-6.5); marker (3-10); ultrapure water;
the preparation method of the S3 instrument comprises the following specific steps:
s301, preparing an isoelectric focusing device: isoelectric focusing devices (Multiphor II multifunctional electrophoresis systems, GE; small cooling water circulation devices, EYELA);
s302, preparing a laser scanning densitometer: Bio-Rad laser scanning densitometer (GS 800);
s303, preparing a horizontal oscillator;
the reagent is prepared by S4, and the specific steps are as follows:
s401, preparing a 30% acrylamide solution: weighing 58.2g of acrylamide and 1.8g of methylene bisacrylamide, dissolving with 150ml of pure water, then fixing the volume to 200ml, filtering by a 0.22 mu m membrane, and storing in the dark at 5 +/-3 ℃;
S402, preparing a stationary liquid: trichloroacetic acid mother liquor: weighing 100g of trichloroacetic acid, adding 100mL of water, mixing, and uniformly mixing before use; 20% trichloroacetic acid: mixing 20mL of mother liquor with 80mL of water (prepared as before use);
s403, preparing a dyeing solution: dyeing stock solution: weighing 125g of ammonium sulfate, and dissolving with 400ml of pure water to obtain solution 1; weighing 1.0G of Coomassie brilliant blue G250, adding 20ml of pure water, stirring, adding 20G of phosphoric acid, mixing with the solution 1 after dissolving, metering the volume to 1000ml, and fully shaking up before use; working dyeing liquid: taking 60ml of dyeing stock solution, mixing with 30ml of methanol uniformly, and carrying out new preparation for use;
S404, preparing 10% APS: 0.1g of sub-packaged ammonium persulfate is taken and dissolved in 1ml of ultrapure water, and the ammonium persulfate is prepared as it is (or stored at 5 +/-3 ℃ for 1 week for use);
s405, preparing an electrode solution: anode solution: measuring 57.6mL of phosphoric acid in 500mL of water, metering to 1000mL of phosphoric acid solution at 0.5mol/L, filtering with a 0.22 mu m membrane, and storing at normal temperature; and (3) negative electrode solution: weighing 8g of sodium hydroxide in 0.2mol/L sodium hydroxide solution, dissolving in 500mL of water, diluting to 1000mL, filtering with 0.22 μm membrane, and storing at normal temperature;
S406, preparing 80% glycerol: measuring 160ml of glycerol, adding pure water to dilute the glycerol to 200ml, filtering the glycerol by using a 0.45-micron membrane, and storing the glycerol at normal temperature;
s407, preparing a decoloring solution: preparing 250ml of 95% ethanol, 80ml of glacial acetic acid and 670ml of ultrapure water;
s408, preparing a dry glue dehydration solution: 200ml of absolute ethyl alcohol, 50ml of glacial acetic acid, 50ml of glycerol and 100ml of ultrapure water are uniformly mixed and the volume is up to 500 ml.
in a fifth embodiment, referring to fig. 1 to 6, a method for detecting isoelectric points of recombinant proteins by isoelectric focusing electrophoresis comprises the following steps:
s1, material preparation: various materials used in the experiment are properly prepared to ensure that the experiment is smoothly carried out;
s2, preparation of reagents: reagents required by the experiment, namely a sample to be detected and a reference substance, need to be prepared in time for the experiment;
s3, preparing an instrument: the experimental instrument is prepared after being cleaned in advance;
s4, preparation of reagents: the reagent which can not be directly used in the experiment is manually prepared for use, so that the smooth operation of the experiment is ensured;
S5, a detection method: mainly takes electrophoresis as a method to test the test;
s6, analyzing and judging the result: the result is judged by comparing the analysis of the reference substance;
the preparation method of the S1 material comprises the following specific steps:
s101, preparing a polypropylene film: PAG fim (polypropylene film, GE healthcare);
s102, manually cutting the electrode strip: the electrode strip is cut manually and can be cut according to the requirement;
s103, manually cutting sample paper: the sample loading paper needs to be cut manually to ensure the smooth sample loading;
s104, manually cutting a dry adhesive film: manually cutting the dry adhesive film for later use;
wherein, the preparation of the S2 reagent comprises the following specific steps:
S201, preparing a sample to be detected and a reference substance: preparing a sample to be detected and a working reference substance for an experiment;
s202, preparing various experimental reagents: preparing acrylamide (electrophoresis grade); methylene bisacrylamide (electrophoresis grade); ammonia persulfate; trichloroacetic acid; ammonium sulfate; phosphoric acid (85%); sodium hydroxide; methanol; absolute ethyl alcohol; coomassie brilliant blue G250; glycerol; glacial acetic acid; a hydrophobic silane; TEMED; ampholytes (pH3.0-10.0, pH4.0-6.5); marker (3-10); ultrapure water;
the preparation method of the S3 instrument comprises the following specific steps:
s301, preparing an isoelectric focusing device: isoelectric focusing devices (Multiphor II multifunctional electrophoresis systems, GE; small cooling water circulation devices, EYELA);
S302, preparing a laser scanning densitometer: Bio-Rad laser scanning densitometer (GS 800);
S303, preparing a horizontal oscillator;
the reagent is prepared by S4, and the specific steps are as follows:
s401, preparing a 30% acrylamide solution: weighing 58.2g of acrylamide and 1.8g of methylene bisacrylamide, dissolving with 150ml of pure water, then fixing the volume to 200ml, filtering by a 0.22 mu m membrane, and storing in the dark at 5 +/-3 ℃;
s402, preparing stationary liquid; trichloroacetic acid mother liquor: weighing 100g of trichloroacetic acid, adding 100mL of water, mixing, and uniformly mixing before use; 20% trichloroacetic acid: mixing 20mL of mother liquor with 80mL of water (prepared as before use);
s403, preparing a dyeing solution: dyeing stock solution: weighing 125g of ammonium sulfate, and dissolving with 400ml of pure water to obtain solution 1; weighing 1.0G of Coomassie brilliant blue G250, adding 20ml of pure water, stirring, adding 20G of phosphoric acid, mixing with the solution 1 after dissolving, metering the volume to 1000ml, and fully shaking up before use; working dyeing liquid: taking 60ml of dyeing stock solution, mixing with 30ml of methanol uniformly, and carrying out new preparation for use;
s404, preparing 10% APS: 0.1g of sub-packaged ammonium persulfate is taken and dissolved in 1ml of ultrapure water, and the ammonium persulfate is prepared as it is (or stored at 5 +/-3 ℃ for 1 week for use);
s405, preparing an electrode solution: anode solution: measuring 57.6mL of phosphoric acid in 500mL of water, metering to 1000mL of phosphoric acid solution at 0.5mol/L, filtering with a 0.22 mu m membrane, and storing at normal temperature; and (3) negative electrode solution: weighing 8g of sodium hydroxide in 0.2mol/L sodium hydroxide solution, dissolving in 500mL of water, diluting to 1000mL, filtering with 0.22 μm membrane, and storing at normal temperature;
s406, preparing 80% glycerol: measuring 160ml of glycerol, adding pure water to dilute the glycerol to 200ml, filtering the glycerol by using a 0.45-micron membrane, and storing the glycerol at normal temperature;
S407, preparing a decoloring solution: preparing 250ml of 95% ethanol, 80ml of glacial acetic acid and 670ml of ultrapure water;
s408, preparing a dry glue dehydration solution: preparing 200ml of absolute ethyl alcohol, 50ml of glacial acetic acid, 50ml of glycerol and 100ml of ultrapure water, uniformly mixing, and fixing the volume to 500 ml;
The S5 inspection method comprises the following specific steps:
s501, assembling a glue making frame: putting a clean glue-making glass plate on clean wiping cloth, uniformly coating 0.5mL of hydrophobic silane on the front surface of a rubber strip glass plate with the mu m, and airing at room temperature; adding 0.5ml of water to one surface of a plane glass plate for wetting, taking a glue making film, sticking a glass plate on a hydrophobic surface, enabling the hydrophobic surface to be tightly attached to the wetted glass plate by using a manual roller press and removing bubbles completely, and cleaning redundant water by using absorbent paper; two glass plates are symmetrically fixed by using a glue-making clamp at the left side and the right side respectively, and the lower side of the glue-making clamp is two glass plates (pre-cooled at 5 +/-3 ℃ when the room temperature is too high);
s502, preparing and filling a glue solution: urea mother liquor: weighing 10.8g of urea, directly adding 17.5mL of ultrapure water to dissolve the urea to 24.6mL, and filtering by a 0.22 mu m membrane for later use; preparing glue and filling: 3.3ml of 30% polyacrylamide solution, 12.3ml of urea mother liquor, 1.2ml of 80% glycerol, 0.275ml of pH4.0-6.5 electrolyte, 0.825ml of pH3.0-10.0 electrolyte and 100 mul of 10% APS, uniformly mixing, adding 20 mul of TEMED, uniformly mixing, sucking the mixed solution by using a 5ml liquid-transferring gun, filling the mixed solution into gaps of assembled glue-making molds, carefully avoiding bubbles (if bubbles are generated, slightly beating a glass plate by hands, discharging bubbles), standing at room temperature for 1.5h, completely solidifying (when the temperature is too low, standing at 25 ℃ for 2.5 h), immediately using or wrapping by using a preservative film, and storing at 5 +/-3 ℃ for 2-3 days;
s503, pre-electrophoresis: opening a switch of the small cooling water circulation device until the temperature is cooled to 16 ℃, then placing 1mL of ultrapure water on an electrophoresis plate, taking glue, symmetrically sticking the glue on 3-13 parts of the electrophoresis plate, and removing bubbles; and (3) placing an electrode strip (shorter than the glue) at the transverse lines of 3 and 13, adding an appropriate amount (about 1 mL) of negative electrode solution in the negative electrode direction until the electrode strip is wetted, and adding an appropriate amount (about 1 mL) of positive electrode solution in the positive electrode direction until the electrode strip is wetted. Covering an electrophoresis tank cover, opening a switch of an electrophoresis power supply device to set 700V, 24mA and 8W, and pre-electrophoresis for 60 min;
S504, sample treatment: dissolving a control sample with 250ml of ultrapure water to obtain a control sample with the concentration of 2 mg/ml; diluting the sample to 2 mg/ml;
s505, spotting: placing a sample paper sheet (cut into a square of about 0.5X1.0cm by using a piece of lens wiping paper) on the gel at a position 6cm away from the cathode, and spotting a Marker; placing a sample loading paper sheet, a sample application sample and a working reference substance on the gel at a position 1cm away from the cathode, wherein the sample application amount is 20 mu g (the volume is 10 mu l);
s506, electrophoresis: loading and electrophoresis: setting 500V, 8mA, 8W, temperature 10-20 ℃ (general 16 ℃), and electrophoresis for 20 min. Removing the sample loading paper sheet after finishing; formal electrophoresis: setting 2200V, 14mA, 18W, 180min +2500V, 14mA, 20W, 10min, and 10-20 ℃ (generally 16 ℃);
s507, dyeing and decoloring: after electrophoresis, taking a film, enabling the rubber surface to face upwards, placing the film in a fixing solution, oscillating and fixing the film for 20min at room temperature and 10rpm, oscillating and cleaning the film for 2min with clear water after the electrophoresis is finished, then oscillating and dyeing the film in a dyeing solution for 1 h until bands are clearly shown (when the temperature in winter is too low, the dyeing time is properly prolonged until the bands are clearly shown), rinsing redundant dyeing solution with clear water when dyeing is stopped, placing the film in clear water, slightly wiping the film with a common paper towel to remove floating color (when wiping, keeping the film soaked in the water to keep moist so as not to wipe the rubber surface), and placing the film in a decoloring solution to remove a background;
s508, glue sweeping: GS800 scanning analysis isoelectric point;
s509, preparing dry glue: placing the film in a small amount of dry glue dewatering liquid, oscillating for 5min, placing on a glass plate, sticking a dry glue film, sealing the periphery with transparent glue, and naturally drying in a fume hood.
the working principle is as follows: the invention provides a method for detecting isoelectric points of isoelectric focusing electrophoresis of recombinant proteins, which comprises the steps of preparing materials, reagents and instruments, reasonably preparing the reagents, scientifically detecting the reagents, strictly analyzing and judging, carrying out isoelectric focusing electrophoresis on the recombinant proteins, and having scientific and reasonable results and convincing effect; preparing the material, namely preparing a polypropylene film; PAG fim (polypropylene film, GE healthcare); the electrode strips, the sample paper and the dry adhesive film are cut manually to ensure that the required materials are cut according to the requirements of the users, so that the experiment can be completed more effectively; the invention further provides for the preparation of said apparatus, preparing isoelectric focusing devices (Multiphor II multifunctional electrophoresis systems, GE; small cooling water circulation devices, EYELA); preparing a laser scanning densitometer; Bio-Rad laser scanning densitometer (GS 800); preparing a horizontal oscillator, cleaning and checking the horizontal oscillator in advance, and preparing for an experiment; the invention further provides reagent preparation, namely, a sample to be detected and a reference substance are prepared; preparing a sample to be detected and a working reference substance for an experiment; preparing various experimental reagents; preparing acrylamide (electrophoresis grade); methylene bisacrylamide (electrophoresis grade); ammonia persulfate; trichloroacetic acid; ammonium sulfate; phosphoric acid (85%); sodium hydroxide; methanol; absolute ethyl alcohol; coomassie brilliant blue G250; glycerol; glacial acetic acid; a hydrophobic silane; TEMED; ampholytes (pH3.0-10.0, pH4.0-6.5); marker (3-10); ultrapure water and the like are prepared in types to ensure that the experiment can be carried out smoothly;
the invention further discloses a reagent for preparing the reagent, which is used for preparing a 30% acrylamide solution; weighing 58.2g of acrylamide and 1.8g of methylene bisacrylamide, dissolving with 150ml of pure water, then fixing the volume to 200ml, filtering by a 0.22 mu m membrane, and storing in the dark at 5 +/-3 ℃; preparing a stationary liquid; trichloroacetic acid mother liquor: weighing 100g of trichloroacetic acid, adding 100mL of water, mixing, and uniformly mixing before use; 20% trichloroacetic acid: mixing 20mL of mother liquor with 80mL of water (prepared as before use); preparing a dyeing solution; dyeing stock solution: weighing 125g of ammonium sulfate, and dissolving with 400ml of pure water to obtain solution 1; weighing 1.0G of Coomassie brilliant blue G250, adding 20ml of pure water, stirring, adding 20G of phosphoric acid, mixing with the solution 1 after dissolving, metering the volume to 1000ml, and fully shaking up before use; working dyeing liquid: taking 60ml of dyeing stock solution, mixing with 30ml of methanol uniformly, and carrying out new preparation for use; preparing 10% APS; 0.1g of sub-packaged ammonium persulfate is taken and dissolved in 1ml of ultrapure water, and the ammonium persulfate is prepared as it is (or stored at 5 +/-3 ℃ for 1 week for use); preparing an electrode solution; anode solution: measuring 57.6mL of phosphoric acid in 500mL of water, metering to 1000mL of phosphoric acid solution at 0.5mol/L, filtering with a 0.22 mu m membrane, and storing at normal temperature; and (3) negative electrode solution: weighing 8g of sodium hydroxide in 0.2mol/L sodium hydroxide solution, dissolving in 500mL of water, diluting to 1000mL, filtering with 0.22 μm membrane, and storing at normal temperature; preparing 80% glycerol; measuring 160ml of glycerol, adding pure water to dilute the glycerol to 200ml, filtering the glycerol by using a 0.45-micron membrane, and storing the glycerol at normal temperature; preparing a decoloring solution; preparing 250ml of 95% ethanol, 80ml of glacial acetic acid and 670ml of ultrapure water; preparing a dry glue dehydrating solution; 200ml of absolute ethyl alcohol, 50ml of glacial acetic acid, 50ml of glycerol and 100ml of ultrapure water are uniformly mixed, the constant volume is up to 500ml, the reagent is prepared in strict proportion and quantity, and the reagent required by the experiment is scientifically and reasonably prepared;
the invention further discloses a detection method, which comprises the following steps of: putting a clean glue-making glass plate on clean wiping cloth, uniformly coating 0.5mL of hydrophobic silane on the front surface of a rubber strip glass plate with the mu m, and airing at room temperature; adding 0.5ml of water to one surface of a plane glass plate for wetting, taking a glue making film, sticking a glass plate on a hydrophobic surface, enabling the hydrophobic surface to be tightly attached to the wetted glass plate by using a manual roller press and removing bubbles completely, and cleaning redundant water by using absorbent paper; two glass plates are symmetrically fixed by using a glue-making clamp at the left side and the right side respectively, and the lower side of the glue-making clamp is two glass plates (pre-cooled at 5 +/-3 ℃ when the room temperature is too high); preparing and filling a glue solution: urea mother liquor: weighing 10.8g of urea, directly adding 17.5mL of ultrapure water to dissolve the urea to 24.6mL, and filtering by a 0.22 mu m membrane for later use; preparing glue and filling: 3.3ml of 30% polyacrylamide solution, 12.3ml of urea mother liquor, 1.2ml of 80% glycerol, 0.275ml of pH4.0-6.5 electrolyte, 0.825ml of pH3.0-10.0 electrolyte and 100 mul of 10% APS, uniformly mixing, adding 20 mul of TEMED, uniformly mixing, sucking the mixed solution by using a 5ml liquid-transferring gun, filling the mixed solution into gaps of assembled glue-making molds, carefully avoiding bubbles (if bubbles are generated, slightly beating a glass plate by hands, discharging bubbles), standing at room temperature for 1.5h, completely solidifying (when the temperature is too low, standing at 25 ℃ for 2.5 h), immediately using or wrapping by using a preservative film, and storing at 5 +/-3 ℃ for 2-3 days; pre-electrophoresis: opening a switch of the small cooling water circulation device until the temperature is cooled to 16 ℃, then placing 1mL of ultrapure water on an electrophoresis plate, taking glue, symmetrically sticking the glue on 3-13 parts of the electrophoresis plate, and removing bubbles; and (3) placing an electrode strip (shorter than the glue) at the transverse lines of 3 and 13, adding an appropriate amount (about 1 mL) of negative electrode solution in the negative electrode direction until the electrode strip is wetted, and adding an appropriate amount (about 1 mL) of positive electrode solution in the positive electrode direction until the electrode strip is wetted. Covering an electrophoresis tank cover, opening a switch of an electrophoresis power supply device to set 700V, 24mA and 8W, and pre-electrophoresis for 60 min; processing a sample; dissolving a control sample with 250ml of ultrapure water to obtain a control sample with the concentration of 2 mg/ml; diluting the sample to 2 mg/ml; sample application: a piece of sample paper (cut into squares of about 0.5X1.0cm with a lens-wiping paper) was placed on the gel at a distance of 6cm from the cathode, and a point Marker (Marker): placing a sample loading paper sheet, a sample application sample and a working reference substance on the gel at a position 1cm away from the cathode, wherein the sample application amount is 20 mu g (the volume is 10 mu l); electrophoresis: loading and electrophoresis: setting 500V, 8mA, 8W, temperature 10-20 ℃ (general 16 ℃), and electrophoresis for 20 min. Removing the sample loading paper sheet after finishing; formal electrophoresis: setting 2200V, 14mA, 18W, 180min +2500V, 14mA, 20W, 10min, and 10-20 ℃ (generally 16 ℃); dyeing and decoloring: after electrophoresis, taking a film, enabling the rubber surface to face upwards, placing the film in a fixing solution, oscillating and fixing the film for 20min at room temperature and 10rpm, oscillating and cleaning the film for 2min with clear water after the electrophoresis is finished, then oscillating and dyeing the film in a dyeing solution for 1 h until bands are clearly shown (when the temperature in winter is too low, the dyeing time is properly prolonged until the bands are clearly shown), rinsing redundant dyeing solution with clear water when dyeing is stopped, placing the film in clear water, slightly wiping the film with a common paper towel to remove floating color (when wiping, keeping the film soaked in the water to keep moist so as not to wipe the rubber surface), and placing the film in a decoloring solution to remove a background; glue sweeping: GS800 scanning analysis isoelectric point; and (3) drying glue: placing the film in a small amount of dry glue dehydration solution, oscillating for 5min, placing on a glass plate, sticking a dry glue film, sealing the periphery with transparent glue, and naturally drying in a fume hood; the test is mainly carried out through electrophoresis so as to achieve the purpose of experiment;
according to the further analysis and judgment result, each sample group must contain at least one reference substance, and each lane should be vertical without diffusion and well separated; the bands of the reference substance are distributed within the range of pI 4.6-6.8, the migration distance of the main components of the sample is consistent with that of the reference substance, and the limiting conditions of the sample and the reference substance are favorable for better comparison.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. an isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein is characterized in that: the method comprises the following steps:
s1, material preparation: various materials used in the experiment are properly prepared to ensure that the experiment is smoothly carried out;
s2, preparation of reagents: reagents required by the experiment, namely a sample to be detected and a reference substance, need to be prepared in time for the experiment;
S3, preparing an instrument: the experimental instrument is prepared after being cleaned in advance;
s4, preparation of reagents: the reagent which can not be directly used in the experiment is manually prepared for use, so that the smooth operation of the experiment is ensured;
s5, a detection method: mainly takes electrophoresis as a method to test the test;
S6, analyzing and judging the result: and (5) judging a result by comparing the reference substance and analyzing.
2. The method for isoelectric focusing electrophoresis (isoelectric point) detection of recombinant proteins according to claim 1, comprising: the preparation method of the S1 material comprises the following specific steps:
s101, preparing a polypropylene film;
S102, manually cutting the electrode strip: the electrode strip is cut manually and can be cut according to the requirement;
s103, manually cutting sample paper: the sample loading paper needs to be cut manually to ensure the smooth sample loading;
s104, manually cutting a dry adhesive film: the dry film was cut manually for later use.
3. The method for isoelectric focusing electrophoresis isoelectric point detection of recombinant protein according to claim 2, characterized in that: the preparation method of the S2 reagent comprises the following specific steps:
S201, preparing a sample to be detected and a reference substance; preparing a sample to be detected and a working reference substance for an experiment;
S202, preparing various experimental reagents: preparing acrylamide; methylene bisacrylamide; ammonia persulfate; trichloroacetic acid; ammonium sulfate; 85% phosphoric acid; sodium hydroxide; methanol; absolute ethyl alcohol; coomassie brilliant blue G250; glycerol; glacial acetic acid; a hydrophobic silane; TEMED; ampholytes (pH3.0-10.0, pH4.0-6.5); marker (3-10); marker; ultrapure water.
4. the method for detecting isoelectric point of recombinant protein according to claim 3, which comprises the steps of: preparing the S3 instrument, wherein the specific steps are as follows:
S301, preparing an isoelectric focusing device;
s302, preparing a laser scanning densitometer: Bio-Rad laser scanning densitometer;
and S303, preparing a horizontal oscillator.
5. the method for isoelectric focusing electrophoresis (isoelectric point) detection of recombinant protein according to claim 4, wherein the reagent is prepared from S4 by the following steps:
s401, preparing a 30% acrylamide solution: weighing 58.2g of acrylamide and 1.8g of methylene bisacrylamide, dissolving with 150ml of pure water, then fixing the volume to 200ml, filtering by a 0.22 mu m membrane, and storing in the dark at 5 +/-3 ℃;
s402, preparing a stationary liquid: trichloroacetic acid mother liquor: weighing 100g of trichloroacetic acid, adding 100mL of water, mixing, and uniformly mixing before use; 20% trichloroacetic acid: mixing 20mL of mother liquor with 80mL of water;
s403, preparing a dyeing solution: dyeing stock solution: weighing 125g of ammonium sulfate, and dissolving with 400ml of pure water to obtain solution 1; weighing 1.0G of Coomassie brilliant blue G250, adding 20ml of pure water, stirring, adding 20G of phosphoric acid, mixing with the solution 1 after dissolving, metering the volume to 1000ml, and fully shaking up before use; working dyeing liquid: taking 60ml of dyeing stock solution, mixing with 30ml of methanol uniformly, and carrying out new preparation for use;
s404, preparing 10% APS; 0.1g of sub-packaged ammonium persulfate is taken and dissolved in 1ml of ultrapure water, and the mixture is prepared as it is used;
s405, preparing an electrode solution: anode solution: measuring 57.6mL of phosphoric acid in 500mL of water, metering to 1000mL of phosphoric acid solution at 0.5mol/L, filtering with a 0.22 mu m membrane, and storing at normal temperature; and (3) negative electrode solution: weighing 8g of sodium hydroxide in 0.2mol/L sodium hydroxide solution, dissolving in 500mL of water, diluting to 1000mL, filtering with 0.22 μm membrane, and storing at normal temperature;
s406, preparing 80% glycerol: measuring 160ml of glycerol, adding pure water to dilute the glycerol to 200ml, filtering the glycerol by using a 0.45-micron membrane, and storing the glycerol at normal temperature;
S407, preparing a decoloring solution: preparing 250ml of 95% ethanol, 80ml of glacial acetic acid and 670ml of ultrapure water;
s408, preparing a dry glue dehydration solution: 200ml of absolute ethyl alcohol, 50ml of glacial acetic acid, 50ml of glycerol and 100ml of ultrapure water are uniformly mixed and the volume is up to 500 ml.
6. The method for isoelectric focusing electrophoresis isoelectric point detection of recombinant protein according to claim 5, characterized in that: the S5 inspection method comprises the following specific steps:
s501, assembling a glue making frame: putting a clean glue-making glass plate on clean wiping cloth, uniformly coating 0.5mL of hydrophobic silane on the front surface of a rubber strip glass plate with the mu m, and airing at room temperature; adding 0.5ml of water to one surface of a plane glass plate for wetting, taking a glue making film, sticking a glass plate on a hydrophobic surface, enabling the hydrophobic surface to be tightly attached to the wetted glass plate by using a manual roller press and removing bubbles completely, and cleaning redundant water by using absorbent paper; the left glass plate and the right glass plate are fixed by glue-making clamps respectively, and the two glass plates are symmetrically fixed by the lower two glue-making clamps;
s502, preparing and filling a glue solution: urea mother liquor: weighing 10.8g of urea, directly adding 17.5mL of ultrapure water to dissolve the urea to 24.6mL, and filtering by a 0.22 mu m membrane for later use; preparing glue and filling: 3.3ml of 30% polyacrylamide solution, 12.3ml of urea mother liquor, 1.2ml of 80% glycerol, 0.275ml of pH4.0-6.5 electrolyte, 0.825ml of pH3.0-10.0 electrolyte and 100 mul of 10% APS, uniformly mixing, adding 20 mul of TEMED, uniformly mixing, sucking the mixed solution by using a 5ml liquid transfer gun, filling the mixed solution into gaps of assembled glue-making molds, ensuring that no bubbles are generated, standing at room temperature for 1.5h, completely solidifying, immediately using or wrapping by using a preservative film, and storing at 5 +/-3 ℃, wherein the preservative film can be used within 2-3 days;
s503, pre-electrophoresis: opening a switch of the small cooling water circulation device until the temperature is cooled to 16 ℃, then placing 1mL of ultrapure water on an electrophoresis plate, taking glue, symmetrically sticking the glue on 3-13 parts of the electrophoresis plate, and removing bubbles; and (3) placing the electrode strips at the transverse lines of 3 and 13, adding a proper amount of negative liquid in the negative direction until the electrode strips are wetted, and adding a proper amount of positive liquid in the positive direction until the electrode strips are wetted. Covering an electrophoresis tank cover, opening a switch of an electrophoresis power supply device to set 700V, 24mA and 8W, and pre-electrophoresis for 60 min;
s504, sample treatment: dissolving a control sample with 250ml of ultrapure water to obtain a control sample with the concentration of 2 mg/ml; diluting the sample to 2 mg/ml;
s505, spotting: placing a sample loading paper sheet on the gel at a position 6cm away from the cathode, and spotting a Marker; placing a sample loading paper sheet, a sample application sample and a working reference substance on the gel at a position 1cm away from the cathode, wherein the sample application amount is 20 mu g;
S506, electrophoresis; loading and electrophoresis: setting 500V, 8mA, 8W, temperature of 10-20 ℃, and electrophoresis for 20 min. Removing the sample loading paper sheet after finishing; formal electrophoresis: setting 2200V, 14mA, 18W, 180min +2500V, 14mA, 20W, 10min and 10-20 ℃ of temperature;
s507, dyeing and decoloring: after electrophoresis, taking a film, enabling the rubber surface to face upwards, placing the film in a fixing solution, oscillating and fixing the film for 20min at room temperature at 10rpm, oscillating and cleaning the film for 2min with clear water after the electrophoresis is finished, oscillating and dyeing the film in a dyeing solution for 1 hour until strips are clearly shown, rinsing redundant dyeing solution with clear water when dyeing is stopped, placing the film in the clear water, slightly wiping the film with a common paper towel to remove floating color, and placing the film in a decoloring solution to remove background;
s508, glue sweeping: GS800 scanning analysis isoelectric point;
s509, preparing dry glue: placing the film in a small amount of dry glue dewatering liquid, oscillating for 5min, placing on a glass plate, sticking a dry glue film, sealing the periphery with transparent glue, and naturally drying in a fume hood.
7. the method for isoelectric focusing electrophoresis (isoelectric point) detection of recombinant proteins according to claim 1, comprising: in the S6 analysis and determination result, each sample group must contain at least one reference substance, and each lane should be vertical without diffusion and well separated; the bands of the reference substance should be distributed within the range of pI 4.6-6.8, and the migration distance of the main components of the sample should be consistent with that of the reference substance.
8. the method for detecting isoelectric point of recombinant protein according to claim 6, which comprises the steps of: in the S502 glue solution preparation and filling, if bubbles are generated, the glass plate is slightly knocked by hands to discharge the bubbles; when the temperature is too low, the mixture is placed in an environment with the temperature of 25 ℃ for solidification for 2.5 hours; in the S503 pre-electrophoresis, 700V, 24mA and 8W are set; during electrophoresis, attention should be paid to the change of relevant parameters such as voltage, and when electrophoresis is finished, the voltage should reach the set upper limit voltage.
9. the method for detecting isoelectric point of recombinant protein according to claim 6, which comprises the steps of: in the S507 dyeing and decoloring, when the temperature is too low in winter, the dyeing time is properly prolonged until the strips are clearly shown; the rubber sheet should be kept wet by soaking in water during wiping so as to avoid rubbing the rubber surface.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1460520A (en) * | 2003-06-03 | 2003-12-10 | 新疆保利达科工贸有限责任公司 | Compound alliin enteric solubility capsule |
| CN1990862A (en) * | 2005-12-28 | 2007-07-04 | 上海华新生物高技术有限公司 | Production method of recombined human alpha1-thymus peptide and preparation thereof |
| CN101168559A (en) * | 2007-09-28 | 2008-04-30 | 西北农林科技大学 | Method for separating and purifying Chinese cabbage differential protein/isozyme |
| CN102288470A (en) * | 2011-07-20 | 2011-12-21 | 中国热带农业科学院热带生物技术研究所 | Coomassie brilliant blue G250 staining method, special staining solution and application thereof |
| CN103257173A (en) * | 2013-02-28 | 2013-08-21 | 青岛农业大学 | Chinese cabbage variety identification and seed purity testing method |
| CN103333867A (en) * | 2013-05-10 | 2013-10-02 | 拉芳家化股份有限公司 | Cationization modified superoxide dismutase and application thereof |
| CN106814122A (en) * | 2015-11-30 | 2017-06-09 | 成都金凯生物技术有限公司 | The improved method that a kind of capillary isoelectric focusing determines recombinant protein isoelectric point |
| CN106932459A (en) * | 2015-12-31 | 2017-07-07 | 深圳翰宇药业股份有限公司 | A kind of polypeptide polymer capillary isoelectric focusing analysis method |
| CN107860814A (en) * | 2017-12-08 | 2018-03-30 | 丽珠集团丽珠制药厂 | A kind of protein isoelectric focusing electrophoresis method and its with adhesive dispenser |
| CN107991368A (en) * | 2017-12-19 | 2018-05-04 | 广东省生物资源应用研究所 | The isoelectric focusing electrophoresis method of hematopoietin |
-
2019
- 2019-10-17 CN CN201910989860.5A patent/CN110554081A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1460520A (en) * | 2003-06-03 | 2003-12-10 | 新疆保利达科工贸有限责任公司 | Compound alliin enteric solubility capsule |
| CN1990862A (en) * | 2005-12-28 | 2007-07-04 | 上海华新生物高技术有限公司 | Production method of recombined human alpha1-thymus peptide and preparation thereof |
| CN101168559A (en) * | 2007-09-28 | 2008-04-30 | 西北农林科技大学 | Method for separating and purifying Chinese cabbage differential protein/isozyme |
| CN102288470A (en) * | 2011-07-20 | 2011-12-21 | 中国热带农业科学院热带生物技术研究所 | Coomassie brilliant blue G250 staining method, special staining solution and application thereof |
| CN103257173A (en) * | 2013-02-28 | 2013-08-21 | 青岛农业大学 | Chinese cabbage variety identification and seed purity testing method |
| CN103333867A (en) * | 2013-05-10 | 2013-10-02 | 拉芳家化股份有限公司 | Cationization modified superoxide dismutase and application thereof |
| CN106814122A (en) * | 2015-11-30 | 2017-06-09 | 成都金凯生物技术有限公司 | The improved method that a kind of capillary isoelectric focusing determines recombinant protein isoelectric point |
| CN106932459A (en) * | 2015-12-31 | 2017-07-07 | 深圳翰宇药业股份有限公司 | A kind of polypeptide polymer capillary isoelectric focusing analysis method |
| CN107860814A (en) * | 2017-12-08 | 2018-03-30 | 丽珠集团丽珠制药厂 | A kind of protein isoelectric focusing electrophoresis method and its with adhesive dispenser |
| CN107991368A (en) * | 2017-12-19 | 2018-05-04 | 广东省生物资源应用研究所 | The isoelectric focusing electrophoresis method of hematopoietin |
Non-Patent Citations (2)
| Title |
|---|
| 王震生: ""蛋白磷酸酶同工酶鉴定新方法的研究"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 霍岩 等: ""载体两性电解质等电聚焦电泳及其在重组蛋白药物研究中的应用"", 《粮食科技与经济》 * |
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