Semi-quantitative method of polymer protein

Abstract

The invention discloses a semi-quantitative method of polymer protein, which comprises the following steps: cross-linking the polymer protein by using formaldehyde to enhance the stability and durability of the polymer protein; meanwhile, the electrophoresis method is improved, and the electrophoresis time and operation steps are saved. Compared with a Native-PAGE method, when the method is used for detecting the polymer protein, a result map with higher resolution can be obtained, the protein spectrum band boundary is more obvious, degradation caused by background miscellaneous bands and the operation process is less, the trailing condition is reduced, and the accuracy of semi-quantitative analysis is improved while the resolution is improved. Meanwhile, the method is simple and convenient to operate, time is saved, and an electrophoresis method which is convenient for a general laboratory to quickly, simply and conveniently carry out semi-quantification on the polymer protein is formed.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a semi-quantitative method for multimeric proteins. Background Art

[0002] In biochemistry and molecular biology research, protein separation and analysis are basic and critical steps, especially for the study of multimeric proteins. Protein polymers and their interactions play a key role in many biological processes, including signal transduction, gene expression, etc. In particular, quantitative detection of multimeric proteins is a key means to measure protein expression levels, which helps to reveal biological processes and disease mechanisms and provide important data for biomedical research.

[0003] In the prior art, the determination of protein content includes protein quantitative detection and protein semi-quantitative detection. There are many methods for protein quantitative detection, including: (1) SEC-HPLC method: This method is suitable for soluble protein aggregates, but it has the limitation that sample dilution may lead to aggregate dissociation. Different proteins require different mobile phases, and mobile phases or high pressure may also induce protein to form aggregates; (2) Light scattering method: including in situ dynamic light scattering and in situ static light scattering, which are used to detect protein aggregates, but the light scattering signal is easily interfered by factors such as bubbles, small particles and impurities in the sample itself, and the instrument equipment is expensive; (3) Analytical ultracentrifugation method: Although this method has a certain degree of accuracy, the operation process includes different speeds. The centrifugation process will damage the multimeric protein to a certain extent. The operation is cumbersome and time-consuming. The purity of the obtained protein is relatively low and the recovery rate is unstable. (4) Bradford method, BCA method and Lowry method: Bradford method is a convenient detection method commonly used in laboratories. The color of Coomassie Brilliant Blue G250 changes after binding with protein, and the absorbance value is proportional to the protein content. However, it is easily affected by SDS detergent when detecting multimeric proteins. The BCA method uses protein to reduce copper sulfate under alkaline conditions and chelate with BCA to form a blue-purple complex. The protein concentration is calculated by measuring the absorbance at 562nm, but the protein is easily affected by chelating agents. The Lowry method uses the peptide bond of protein in alkaline solution to bind to Cu 2+Chelation forms a protein-copper complex and produces a blue compound. The depth of blue is linearly related to the protein concentration, but its specificity is poor. Moreover, these three methods can only detect protein concentration and cannot distinguish whether the detected protein is a multimeric protein. The semi-quantitative method for protein is mainly polyacrylamide gel electrophoresis, including denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and non-denaturing polyacrylamide gel electrophoresis (Native-PAGE). Although SDS-PAGE can effectively separate proteins according to their molecular weight and has the characteristics of high resolution, it has some limitations when detecting multimeric proteins. Since SDS-PAGE usually needs to be performed under denaturing conditions, which will destroy the natural structure and function of multimeric proteins and further lead to the degradation of multimeric proteins, SDS-PAGE cannot be used for the detection of multimeric proteins. Native-PAGE does not contain denaturants such as SDS and mercaptoethanol, so it will not affect protein activity and is widely used in the study of dimers or multimeric proteins. However, the separation efficiency and resolution of Native-PAGE detection results still need to be improved, and Native-PAGE is particularly cumbersome during operation, which will lead to protein degradation and tailing. Therefore, it is of great significance to provide a semi-quantitative detection method for polymers with high resolution, simple operation, less protein degradation during the process, and no tailing to overcome the limitations of existing technologies and meet the higher requirements for polymer protein analysis in biochemical and molecular biological research. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a semi-quantitative method for multimeric proteins.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a semi-quantitative method for multimeric protein, comprising the following steps:

[0007] (1) Pretreatment of polymer protein electrophoresis samples:

[0008] S1. Performing a cross-linking reaction on the sample to be tested, and terminating the cross-linking reaction after the reaction is completed;

[0009] S2. extracting the protein in the cross-linked sample to be tested, and adding a loading buffer without a denaturant;

[0010] S3. Heat at 36-38°C for 5-10 min or at 95-100°C for 3-5 min to obtain a pretreated protein sample;

[0011] (2) Electrophoresis detection of pre-treated protein samples:

[0012] S4. Electrophoresis of the pretreated protein sample in step S3 is performed using a tris-glycine system electrophoresis gel and an electrophoresis solution composed of water, tris-glycine, glycine and sodium dodecyl sulfate; electrophoresis conditions: room temperature, first 70-90V, 20-40min, then 100-130V until the end of electrophoresis;

[0013] S5. Analyze the electrophoresis results.

[0014] The present invention first uses a cross-linking agent to cross-link proteins in the sample to be tested, and chemical bonding occurs between proteins, thereby fixing the multimeric protein structure, reducing the degradation of the multimeric structure during pretreatment and electrophoresis, and using a stopper to stop the cross-linking reaction. Further, adding a loading buffer without a denaturant to the protein sample after extraction and cross-linking and then heating it can avoid the dissociation of the multimeric protein caused by the denaturant. The heating reaction can also dissociate the non-specific binding protein introduced when the cross-linking agent cross-links the protein, and will not cause the dissociation of the multimeric protein. Since the stability and durability of the protein are increased after cross-linking, during the electrophoresis operation, a gel without a denaturant and an electrophoresis solution containing a denaturant can be used at room temperature to electrophorese the multimeric protein. The gel without a denaturant will not cause the dissociation of the multimeric protein, and the electrophoresis solution containing a denaturant will balance the charge of the multimeric protein, so that the multimeric protein is separated only according to the molecular weight of the multimeric protein during the electrophoresis process. Normal temperature operation can shorten the electrophoresis time and simplify the electrophoresis operation. After the electrophoresis is completed, the multimeric protein is quantitatively judged.

[0015] Furthermore, the crosslinking agent used in the crosslinking reaction in step S1 is formaldehyde. Formaldehyde is a commonly used crosslinking agent, which has the characteristics of small molecules and easy penetration of cell membranes. It can crosslink and fix proteins and DNA in a natural state before cells respond to stress. Formaldehyde reacts chemically with amino and carboxyl groups in proteins to form new chemical structures through alkylation and condensation reactions, thereby enhancing the stability and durability of proteins.

[0016] Furthermore, the final concentration of the formaldehyde is 1-2%.

[0017] Preferably, the final concentration of formaldehyde is 1%. When the formaldehyde concentration is too low, it cannot cause protein cross-linking; but when the formaldehyde concentration is too high, it may cause changes in cellular protein levels and may produce false positive or false negative research results.

[0018] Furthermore, the terminating agent used to terminate the cross-linking reaction in step S2 is glycine. This is because during the process of formaldehyde cross-linking proteins, formaldehyde reacts with amino groups in the protein to form a cross-linked structure. When glycine is added, glycine, as an amino acid, also contains amino groups, so it will compete with formaldehyde to terminate the cross-linking process.

[0019] Furthermore, the final concentration of glycine is 0.12-0.13 M. When the concentration of glycine is too low, the cross-linking reaction of formaldehyde cannot be completely terminated, resulting in the continuation of the cross-linking process, which affects the accuracy of subsequent experimental results; when the concentration of glycine is too high, although the cross-linking reaction can be terminated more effectively, the excessively high concentration may bring unnecessary waste, and may even have other unknown effects on the experimental system, interfering with the accuracy of subsequent tests.

[0020] Furthermore, in step S3, the heating is performed at 37° C. for 10 min or 100° C. for 5 min. Because formaldehyde can also cross-link some non-specific, randomly occurring proteins, affecting the subsequent detection results, but the cross-linking of non-specific proteins is very unstable, high temperature treatment will not lead to the dissociation of multimeric proteins, but will affect the non-specific binding proteins introduced when the formaldehyde cross-links the proteins.

[0021] Furthermore, the gel in step S4 consists of a concentration gel and a separation gel.

[0022] Furthermore, the formula of the separation gel is 44.4% to 45.4% ultrapure water, 26.7% to 32.8% of 30% acrylamide-methylenebisacrylamide solution, 21.9% to 26.7% of tris(hydroxymethyl)aminomethane hydrochloride) (Tris-HCl) buffer with pH = 8.8, 0.9% to 1.2% of 10% ammonium persulfate solution, and 0.07% to 0.09% of N,N,N',N'-tetramethylethylenediamine (TEMED).

[0023] Furthermore, the formula of the concentrated gel is 56.5% to 58.7% ultrapure water, 16.4% to 16.9% of 30% acrylamide-methylenebisacrylamide solution, 23.5% to 25.4% of tris(hydroxymethyl)aminomethane hydrochloride buffer with pH=6.8, 1.1% to 1.2% of 10% ammonium persulfate solution, and 0.1% to 0.2% of N,N,N',N'-tetramethylethylenediamine.

[0024] Furthermore, in step S4, the mass volume ratio (mL:g) of water, tris (hydroxymethylaminomethane), glycine and sodium dodecyl sulfate (SDS) is 730-770:2.5-3:10.5-11:0.7-0.8. The significance of adding SDS at this time is to provide charge and balance the difference in charge in the multimeric protein, so that the multimeric protein can be distinguished according to the molecular weight during electrophoresis detection, and the multimeric protein will not be dissociated during electrophoresis.

[0025] Furthermore, the analysis method in step S5 is silver staining, Coomassie brilliant blue staining or antibody detection after electrotransfer.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a semi-quantitative method for multimeric proteins. The present invention first uses a cross-linking agent to cross-link proteins in a sample to be tested, so that the proteins are chemically bonded, thereby fixing the structure of the multimeric protein, reducing the degradation of the multimeric protein during pretreatment and electrophoresis, and using a stopper to stop the cross-linking reaction. Further, adding a loading buffer without a denaturant to the protein sample after extraction and cross-linking and then heating it can avoid the dissociation of the multimeric protein caused by the denaturant. The heating reaction can also dissociate the non-specific binding protein introduced when the cross-linking agent cross-links the protein, and will not cause the dissociation of the multimeric protein. Since the stability and durability of the protein are increased after cross-linking, during the electrophoresis operation, a gel without a denaturant and an electrophoresis solution containing a denaturant can be used at room temperature to electrophoresed the multimeric protein. The gel without a denaturant will not cause the dissociation of the multimeric protein, and the electrophoresis solution containing a denaturant will balance the charge of the multimeric protein, so that the multimeric protein is separated only according to the molecular weight during the electrophoresis process. Normal temperature operation can shorten the electrophoresis time and simplify the electrophoresis operation. After the electrophoresis is completed, the multimeric protein is quantitatively determined. When the method of the present invention is used to detect multimeric proteins, a higher resolution result spectrum can be obtained compared to the existing Native-PAGE method, the protein band boundary is more obvious, the background band is less, the degradation caused by the operation process is also less, and there will be no tailing. While improving the resolution, the accuracy of semi-quantitative analysis is increased. At the same time, the method of the present invention is highly operable, simple to operate, low cost, and time-saving, forming a convenient general laboratory for fast and easy semi-quantitative electrophoresis of multimeric proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 The protein electrophoresis bands are obtained by using the polymer electrophoresis method of the present invention.

[0030] Figure 2 The protein electrophoresis bands were obtained using the Native-PAGE method.

[0031] Figure 3 The protein electrophoresis bands are obtained by using the electrophoresis method of the present invention without using formaldehyde cross-linking. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] Example 1 Semi-quantitative method for detecting multimeric proteins of the present invention

[0035] (1) Cross-linking: Add 37% formaldehyde to the culture dish containing the culture medium according to the volume of the culture medium in the culture dish, so that the final concentration of formaldehyde is 1%, shake well, and let it stand at room temperature for 10 minutes. The principle of this step is to use formaldehyde to chemically bind between proteins, thereby fixing the polymer protein structure and reducing the degradation of the polymer structure during pre-treatment and filming.

[0036] (2) Stop cross-linking: Add 1.25 M glycine solution to the dish until the final concentration of glycine is 0.125 M, shake gently, and let stand at room temperature for 5 minutes.

[0037] (3) Scrape off cells: Gently wash the cells 2-3 times with cold 1× PBS, dry the remaining liquid and place on ice. Add 35 μL IP lysis buffer to each small dish, gently scrape off the cells in the dish with a cell scraper, and transfer to an EP tube.

[0038] (4) Collect and prepare samples: Place the EP tube on ice for 20 minutes to fully lyse. Centrifuge at 4°C, 12,000 rpm, for 15 minutes, and collect the supernatant. After quantifying the sample, add 5× non-denaturing electrophoresis loading buffer to 1× according to the volume. Heat at 37°C for 10 minutes.

[0039] (5) Prepare gel: Put a 1.5 mm glass plate and a 1 mm glass plate together, and fix the 1 mm glass plate to the gel-making rack with the glass plate facing outward. Prepare different concentrations of Tris-glycine electrophoresis gel as needed. Add 3.08 mL of ultrapure water, 2.48 mL of 30% acrylamide-methylenebisacrylamide solution, 1.88 mL of pH 8.8 Tris-HCl buffer, 75 μL of 10% ammonium persulfate solution, and 5 μL of TEMED to a beaker, shake well, and add to the glass plate. After adding, add a layer of ultrapure water on top of the gel to flatten the gel and let it stand. When a clear dividing line appears and the lower layer of gel solidifies, pour off the upper layer of water and carefully absorb the water on top of the gel from the side with filter paper. Add 2.14 mL of ultrapure water, 0.64 mL of 30% acrylamide-□methylenebisacrylamide solution, 0.94 mL of Tris-HCl buffer at pH 6.8, 45 μL of 10% ammonium persulfate solution, and 5 μL of TEMED to the beaker, shake well, add to the glass plate until it is full, absorb the bubbles, and immediately insert the appropriate hole comb. Let it stand until the upper layer of gel solidifies. You can also use commercial gradient precast gels without SDS.

[0040] (6) Prepare electrophoresis solution: Add 750 mL of ultrapure water, 2.77 g of Tris, 10.8 g of glycine, and 0.75 g of SDS into a beaker and stir until there is no precipitation.

[0041] (7) Remove the solidified gel and place it in an electrophoresis tank. Add electrophoresis buffer, load the sample, and perform electrophoresis. The electrophoresis conditions are room temperature, 70V, and about 30 minutes until the loading buff reaches the boundary between the upper and lower gel layers. Then switch to 110V and perform electrophoresis until the target molecular weight protein can be separated.

[0042] (8) After electrophoresis, the gel is removed and, if necessary, the samples are electrophoresed and incubated with antibodies (proteintech, PFKP Polyclonalantibody) for detection.

[0043] The results are as follows Figure 1 As shown, the multimeric protein detected by the present invention is a tetrameric protein - platelet isoform of phosphofructokinase (PFKP), with a molecular weight of 300 kDa, the uppermost band in the electrophoresis is the target band, and the lower band is a monomeric protein with a molecular weight of 75 kDa. The detection result is accurate and has high resolution.

[0044] Comparative Example 1: Detection of multimeric proteins by Native-PAGE

[0045] (1) Scrape off the cells: discard the culture medium and wash the cells 2-3 times with pre-cooled 1× PBS solution. Aspirate the remaining liquid and place on ice. Add 35 μL IP lysis buffer to each small dish, gently scrape off the cells in the dish with a cell scraper, and transfer to an EP tube.

[0046] (2) Collect and prepare samples: Place the EP tube on ice for 20 minutes to fully lyse. Centrifuge at 4°C, 12,000 rpm, for 15 minutes, and collect the supernatant. After quantifying the sample, add 5× non-denaturing electrophoresis loading buffer to 1× according to the volume.

[0047] (3) Prepare gel: Put a 1.5 mm glass plate and a 1 mm glass plate together, and fix the 1 mm glass plate to the gel-making rack with the side facing outward. Prepare Tris-glycine electrophoresis gel of different concentrations as needed. Add 3.08 mL of ultrapure water, 2.48 mL of 30% acrylamide-methylenebisacrylamide solution, 1.88 mL of pH 8.8 Tris-HCl buffer, 75 μL of 10% ammonium persulfate solution, and 5 μL of TEMED to a beaker, shake well, and add to the glass plate. After adding, add a layer of ultrapure water on top of the gel to flatten the gel and let it stand. When a clear dividing line appears and the lower layer of gel solidifies, pour off the upper layer of water and carefully absorb the water on top of the gel from the side with filter paper. Add 2.14 mL of ultrapure water, 0.64 mL of 30% acrylamide-□methylenebisacrylamide solution, 0.94 mL of Tris-HCl buffer at pH 6.8, 45 μL of 10% ammonium persulfate solution, and 5 μL of TEMED to the beaker, shake well, add to the glass plate until it is full, absorb the bubbles, and immediately insert the appropriate hole comb. Let it stand until the upper layer of gel solidifies. Commercial gradient precast gels without SDS can also be used.

[0048] (5) Prepare electrophoresis solution: Add 750 mL of ultrapure water, 2.77 g of Tris, and 10.8 g of glycine to a beaker and stir until there is no precipitation.

[0049] (6) Remove the solidified gel and place it in an electrophoresis tank. Add electrophoresis solution, load the sample, and perform electrophoresis. The electrophoresis conditions are ice bath, 40 mA constant voltage, and electrophoresis for about 3-4 hours until the bands are completely separated. Keep the temperature low during electrophoresis.

[0050] (7) After electrophoresis, the gel is removed and, if necessary, the samples are electrophoresed and incubated with antibodies (proteintech, PFKP Polyclonalantibody) for detection.

[0051] The results are as follows Figure 2 As shown, the multimeric protein PFKP is a tetramer with a molecular weight of 300 kDa. The uppermost band in the electrophoresis diagram is the target band. It can be observed that compared with the detection method of the present invention, the target band in the detection result of the Native-PAGE method is tailing-shaped and is more severely degraded.

[0052] Comparative Example 2: No formaldehyde fixation but detection of multimeric protein by the electrophoresis method of the present invention

[0053] (1) Scrape off cells: Gently wash the cultured cells 2-3 times with cold 1× PBS, dry the remaining liquid and place on ice. Add 35 μL IP lysis buffer to each small dish, gently scrape off the cells in the dish with a cell scraper, and transfer to an EP tube.

[0054] (2) Collect and prepare samples: Place the EP tube on ice for 20 minutes to fully lyse. Centrifuge at 4°C, 12,000 rpm, for 15 minutes, and collect the supernatant. After quantifying the sample, add 5× non-denaturing electrophoresis loading buffer to 1× according to the volume. Heat at 37°C for 10 minutes.

[0055] (3) Prepare gel: Put a 1.5 mm glass plate and a 1 mm glass plate together, and fix the 1 mm glass plate to the gel-making rack with the side facing outward. Prepare Tris-glycine electrophoresis gel of different concentrations as needed. Add 3.08 mL of ultrapure water, 2.48 mL of 30% acrylamide-methylenebisacrylamide solution, 1.88 mL of pH 8.8 Tris-HCl buffer, 75 μL of 10% ammonium persulfate solution, and 5 μL of TEMED to a beaker, shake well, and add to the glass plate. After adding, add a layer of ultrapure water on top of the gel to flatten the gel and let it stand. When a clear dividing line appears and the lower layer of gel solidifies, pour off the upper layer of water and carefully absorb the water on top of the gel from the side with filter paper. Add 2.14 mL of ultrapure water, 0.64 mL of 30% acrylamide-□methylenebisacrylamide solution, 0.94 mL of Tris-HCl buffer at pH 6.8, 45 μL of 10% ammonium persulfate solution, and 5 μL of TEMED to the beaker, shake well, add to the glass plate until it is full, absorb the bubbles, and immediately insert the appropriate hole comb. Let it stand until the upper layer of gel solidifies. You can also use commercial gradient precast gels without SDS.

[0056] (4) Prepare electrophoresis solution: Add 750 mL of ultrapure water, 2.77 g of Tris, 10.8 g of glycine, and 0.75 g of SDS to a beaker and stir until there is no precipitation.

[0057] (5) Remove the solidified gel and place it in an electrophoresis tank. Add electrophoresis solution, load the sample, and perform electrophoresis. The electrophoresis conditions are room temperature, 70V, and about 30 minutes until the loading buff reaches the boundary between the upper and lower gel layers. Then switch to 110V and perform electrophoresis until the target molecular weight protein can be separated.

[0058] (6) After electrophoresis, the gel is removed and, if necessary, the samples are electrophoresed and incubated with antibodies (proteintech, PFKP Polyclonalantibody) for detection.

[0059] The results are as follows Figure 3 As shown, the multimeric protein PFKP was completely dissociated during normal electrophoresis without formaldehyde fixation.

[0060] In summary, the electrophoresis method for semi-quantitative detection of multimeric proteins provided by the present invention can obtain a result spectrum with higher resolution, more obvious protein band boundaries, fewer background bands, less degradation caused by the operation process, and reduced tailing, thereby improving the accuracy of semi-quantitative analysis while improving the resolution. At the same time, the method of the present invention is simple to operate, low in cost, and saves time.

Claims

1. A semi-quantitative method for multimeric proteins, characterized in that: The following steps are involved: (1) Pretreatment of polymer protein electrophoresis samples: S1. Performing a cross-linking reaction on the sample to be tested, and terminating the cross-linking reaction after the reaction is completed; S2. extracting the protein in the cross-linked sample to be tested, and adding a loading buffer without a denaturant; S3. Heat at 36-38°C for 5-10 min or at 95-100°C for 3-5 min to obtain a pretreated protein sample; (2) Electrophoresis detection of pre-treated protein samples: S4. Electrophoresis of the pretreated protein sample in step S3 is performed using a tris-glycine system electrophoresis gel and an electrophoresis solution composed of water, tris-glycine, glycine and sodium dodecyl sulfate; electrophoresis conditions: room temperature, first 70-90V, 20-40min, then 100-130V until the end of electrophoresis; S5. Analyze the electrophoresis results.

2. The method according to claim 1, characterized in that: The cross-linking agent used in the cross-linking reaction in step S1 is formaldehyde.

3. The method according to claim 2, characterized in that: The final concentration of the formaldehyde is 1-2%.

4. The method according to claim 1, characterized in that: The terminating agent used to terminate the cross-linking reaction in step S1 is glycine.

5. The method according to claim 4, characterized in that: The final concentration of the glycine is 0.12-0.13M.

6. The method according to claim 1, characterized in that: The gel in step S4 consists of a concentration gel and a separation gel.

7. The method according to claim 6, characterized in that: The separation gel has a formula of 44.4% to 45.4% ultrapure water, 26.7% to 32.8% of a 30% acrylamide-methylenebisacrylamide solution, 21.9% to 26.7% of a tris(hydroxymethyl)aminomethane hydrochloride) buffer solution with a pH value of 8.8, 0.9% to 1.2% of a 10% ammonium persulfate solution, and 0.07% to 0.09% of N,N,N',N'-tetramethylethylenediamine.

8. The method according to claim 6, characterized in that: The formula of the concentrated gel is 56.5% to 58.7% ultrapure water, 16.4% to 16.9% of 30% acrylamide-methylenebisacrylamide solution, 23.5% to 25.4% of tris(hydroxymethyl)aminomethane hydrochloride buffer solution with pH=6.8, 1.1% to 1.2% of 10% ammonium persulfate solution, and 0.1% to 0.2% of N,N,N',N'-tetramethylethylenediamine.

9. The method according to claim 1, characterized in that: In the step S4, the mass volume ratio of water, tris(hydroxymethyl)aminomethane, glycine and sodium dodecyl sulfate is 730-770:2.5-3:10.5-11:0.7-0.

8.

10. The method according to claim 1, characterized in that: The analysis method in step S5 is silver staining, Coomassie brilliant blue staining or antibody detection after electrotransfer.

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