Composition for preparing polyacrylamide gel, polyacrylamide gel as well as preparation method and application of polyacrylamide gel

Abstract

The invention provides a composition for preparing polyacrylamide gel, the polyacrylamide gel as well as a preparation method and application of the polyacrylamide gel. The composition comprises a separation glue solution and a concentration glue solution, and the separation glue solution is prepared from the following components in parts by volume: 1 to 1.5 parts of 1.2 to 1.8 mol / L Tris-HCl buffer solution, 1.8 to 2.2 parts of acrylamide solution with the concentration of 20 to 40 weight percent, 0.03 to 0.08 part of lauryl sodium sulfate solution with the concentration of 5 to 15 weight percent, 0.03 to 0.08 part of ammonium persulfate solution with the concentration of 5 to 15 weight percent, 0.001 to 0.006 part of tetramethylethylenediamine and 1.5 to 2.2 parts of water; the concentrated glue solution is prepared from the following components in parts by volume: 0.24 to 0.32 part of an acrylamide solution with the concentration of 20 to 40 weight percent, 0.018 to 0.022 part of a lauryl sodium sulfate solution with the concentration of 5 to 15 weight percent, 0.018 to 0.022 part of an ammonium persulfate solution with the concentration of 5 to 15 weight percent, 0.0018 to 0.0022 part of tetramethylethylenediamine, 0.45 to 0.55 part of a Tris-HCL buffer solution with the concentration of 0.2 to 0.8 mol / L and 1.0 to 1.4 parts of water. The polyacrylamide gel prepared from the composition has high separation degree on proteins with similar molecular weights, and can more accurately detect the stripe distribution of the proteins.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials. Specifically, the present invention relates to a composition for preparing polyacrylamide gel, polyacrylamide gel, and its preparation method and use. Background Art

[0002] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a commonly used electrophoresis technique for separating proteins using polyacrylamide gel as a supporting medium. The Chinese Pharmacopoeia has specific records on the formula of polyacrylamide gel for electrophoresis. Using the formula in the pharmacopoeia can separate proteins with different molecular weights and show different protein bands, but it cannot achieve good separation effects for proteins with very close molecular weights.

[0003] Pertussis is a highly contagious severe acute respiratory infectious disease caused by Bordetella pertussis. It is highly infectious and the general population is susceptible, especially infants and young children. It is one of the major infectious diseases that seriously threaten human health.

[0004] Bordetella pertussis can produce many virulence factors, and these virulence factors can undergo phenotypic changes due to changes in environmental conditions, and the expression levels of virulence factors are also different. These virulence factors include various bioactive substances such as pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), heat-labile enterotoxin (ET), heat-stable enterotoxin (HLT), tracheal cytotoxin (TCT), and adenylate cyclase toxin (ACT). When detecting proteins, the classical SDS-PAGE detection method cannot achieve the separation of proteins with very close molecular weights, thus affecting product quality. Summary of the Invention

[0005] The inventors of the present invention have found through a large number of studies that pertussis toxin (PT) is a multi-subunit protein with a total molecular weight of 105KDa. Subunit S1 is 28KDa, subunit S2 is 23KDa, subunit S3 is 22KDa, subunit S4 is 11.7KDa, and subunit S5 is 9.3KDa. The molecular weights of each subunit are very close. Pertussis adhesin (PRN) is 69KDa, and filamentous hemagglutinin (FHA) is 220KDa. These two proteins are extremely prone to degradation and aggregation, and their molecular weight bands are distributed from 90KDa to 250KDa. For the detection of the above proteins, the classical SDS-PAGE formula cannot achieve good separation.

[0006] In view of the above problems, the object of the present invention is to provide a composition for preparing polyacrylamide gel, the polyacrylamide gel, and its preparation method and use. The polyacrylamide gel prepared by using the composition of the present invention has a high resolution for proteins with similar molecular weights, and can more accurately detect the band distribution of proteins.

[0007] Definition:

[0008] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the definition of terms in this field, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel).

[0009] Although the present invention shows numerical ranges and parameter approximations in a broad scope, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently necessarily contains certain errors, which are caused by the standard deviations existing in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "2 to 40" recited should be considered to include any and all sub-ranges between the minimum value of 2 and the maximum value of 40 (including the endpoints), that is to say, all sub-ranges starting from the minimum value of 2 or greater, such as 2 to 6.1, and sub-ranges ending with the maximum value of 40 or less, such as 5.5 to 40. In addition, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0010] The term "or" used herein can be used interchangeably with the term "and / or", unless the context clearly indicates otherwise.

[0011] The above object of the present invention is achieved by providing the following technical solutions:

[0012] On the one hand, the present invention provides a composition for preparing polyacrylamide gel, which comprises a separating gel solution and a stacking gel solution. The separating gel solution comprises: in terms of volume parts, 1 - 1.5 parts of 1.2 - 1.8 mol / L Tris-HCL buffer solution, 1.8 - 2.2 parts of 20 - 40 wt% acrylamide solution, 0.03 - 0.08 parts of 5 - 15 wt% SDS (sodium dodecyl sulfate) solution, 0.03 - 0.08 parts of 5 - 15 wt% APS (ammonium persulfate) solution, 0.001 - 0.006 parts of TEMED (tetramethylethylenediamine), and 1.5 - 2.2 parts of water;

[0013] The concentrated gel solution contains: 0.24 - 0.32 parts of 20 - 40 wt% acrylamide solution by volume, 0.018 - 0.022 parts of 5 - 15 wt% SDS solution, 0.018 - 0.022 parts of 5 - 15 wt% APS solution, 0.0018 - 0.0022 parts of TEMED, 0.45 - 0.55 parts of 0.2 - 0.8 mol / L Tris-HCL buffer, and 1.0 - 1.4 parts of water.

[0014] Preferably, the separating gel solution contains: 1.2 - 1.3 parts of 1.5 mol / L Tris-HCL buffer by volume, 1.9 - 2.1 parts of 30 wt% acrylamide solution, 0.04 - 0.06 parts of 10 wt% SDS solution, 0.04 - 0.06 parts of 10 wt% APS solution, 0.002 - 0.005 parts of TEMED, and 1.7 - 2 parts of water.

[0015] Preferably, the concentrated gel solution contains: 0.26 - 0.3 parts of 30 wt% acrylamide solution by volume, 0.019 - 0.021 parts of 10 wt% SDS solution, 0.019 - 0.021 parts of 10 wt% APS solution, 0.0019 - 0.0021 parts of TEMED, 0.48 - 0.52 parts of 0.5 mol / L Tris-HCL buffer, and 1.1 - 1.3 parts of water.

[0016] Preferably, the total concentration of solutes in the separating gel solution is 10 - 15 wt%, preferably 11 - 12 wt%.

[0017] Preferably, the total concentration of solutes in the concentrated gel solution is 3 - 5 wt%, preferably 3.9 - 4.6 wt%.

[0018] Preferably, the volume ratio of the separating gel solution to the concentrated gel solution in the composition is 1 - 4:1, preferably 2 - 3:1.

[0019] On the other hand, the present invention provides a polyacrylamide gel prepared from the composition of the present invention.

[0020] On yet another aspect, the present invention provides a method for preparing a polyacrylamide gel from the composition of the present invention, the method comprising the following steps:

[0021] (1) Sequentially add the Tris-HCL buffer, acrylamide solution, SDS solution, APS solution, TEMED, and water in the formula amount of the separating gel solution to a container, mix evenly to obtain a separating gel solution, add the separating gel solution to a gel-making plate, add ultrapure water for liquid sealing, let stand, and remove the ultrapure water after the separating gel solution solidifies to obtain a separating gel layer;

[0022] (2) Add acrylamide solution, SDS solution, APS solution, TEMED, Tris-HCL buffer solution and water in the container in sequence according to the formulation amount of the stacking gel solution, mix evenly to obtain the stacking gel solution, add the stacking gel solution to the upper layer of the separating gel layer in the gel plate, insert a comb, let it stand still, and after the stacking gel solution solidifies, obtain a polyacrylamide gel.

[0023] Preferably, the method further comprises the following step: before step (1), check the leakage of the gel plate.

[0024] Preferably, in step (1), let it stand still for 10 - 30 minutes, preferably 20 minutes.

[0025] Preferably, in step (2), let it stand still for 10 - 30 minutes, preferably 20 minutes.

[0026] On the other hand, the present invention provides a polyacrylamide gel kit, which comprises a composition for preparing polyacrylamide gel according to the present invention.

[0027] On the other hand, the present invention provides a method for detecting polyacrylamide gel electrophoresis. The polyacrylamide gel is prepared from the composition for preparing polyacrylamide gel according to the present invention or the polyacrylamide gel kit according to the present invention. The method comprises the following steps:

[0028] (1) Load the protein sample into the gel wells of the polyacrylamide gel and perform electrophoresis;

[0029] (2) After electrophoresis, stain and decolorize the polyacrylamide gel in sequence until the reaction bands are clear, and scan and save the reaction results.

[0030] Preferably, in step (1), the loading amount of the protein sample is 5 - 10 μg, preferably 5 μg.

[0031] Preferably, the method for detecting polyacrylamide gel electrophoresis is used to detect the molecular weight of proteins.

[0032] Preferably, the protein is selected from one or more of pertussis toxin, pertussis adhesin and filamentous hemagglutinin.

[0033] The present invention has at least the following beneficial effects:

[0034] The polyacrylamide gel prepared by using the composition of the present invention has a high resolution for proteins with similar molecular weights, and can more accurately detect the band distribution of proteins.

[0035] Specifically, the polyacrylamide gel prepared using the composition of the present invention is used for electrophoresis separation of pertussis toxin PT protein. The bands of subunits S1, S2, S3, S4, and S5 of this protein are clearly visible, and the resolution is relatively high. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, where:

[0037] Figure 1 FIG. is the electrophoresis result diagram of the polyacrylamide gel prepared using Formula 1 of the present invention;

[0038] Figure 2 is the electrophoresis result diagram of the polyacrylamide gel prepared using Formula 2 of the present invention;

[0039] Figure 3 is the electrophoresis result diagram of the polyacrylamide gel prepared using the pharmacopoeia formula;

[0040] Figure 4 is the electrophoresis result diagram of the commercially available polyacrylamide gel. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention will be further described in detail below in conjunction with the specific embodiments. The examples given are only for clarifying the present invention and not for limiting the scope of the present invention.

[0042] Unless otherwise specified, the raw materials used in the present invention are all commercially available conventional products.

[0043] Among them, for the preparation method of the purified sample of pertussis toxin PT protein, refer to Section 1 of the "Materials and Methods" part in the following literature: Liang Jiangli, Ma Yan, Gu Qin, etc., Detection of the purity of component acellular DTaP vaccine by size exclusion high performance liquid chromatography [J], Chinese Journal of Vaccines and Immunization, 2019, 25(3): 5.

[0044] 1.5 mol / L Tris-HCL buffer (pH 8.8) and 0.5 mol / L Tris-HCL buffer (pH 6.8) are purchased from Beyotime Biotechnology Co., Ltd.

[0045] 30% acrylamide solution is purchased from Thermo Fisher Scientific, and the mass ratio of monomer acrylamide to methylene bisacrylamide is 29:1.

[0046] Example 1 Preparation and Electrophoresis of Polyacrylamide Gel

[0047] 1. Sample Preparation

[0048] Take 100 μL of the purified pertussis toxin PT protein sample and load it into a 1 mL centrifuge tube.

[0049] 2. Sample treatment

[0050] Reduction treatment: Pipette 40 μL of the sample, add 10 μL of 5x reducing bromophenol blue buffer, heat-denature at 100 °C in a metal bath for 5 min, and centrifuge quickly for 10 seconds for standby.

[0051] 3. Preparation of polyacrylamide gel

[0052] (1) Mold preparation: Assemble the gel-making rack. Take a set of glass plates (one thick and one thin), and note the thickness of the glass plate sandwich (i.e., the thickness of the gel). Insert a set of glass plates into the plastic clip, place it on a clean table with the flat side facing down, ensure the bottoms of the two glass plates are flush, then clamp and fix it on the gel-making rack. Place the bottom of the glass plates on a rubber pad (the purpose is to seal the bottoms of the two glass plates to form a container that can hold the gel).

[0053] (2) Leakage inspection: Fill the gaps between the glass plates with pure water, let it stand for 20 min, and observe whether the entire gel-making mold leaks. If there is leakage, check whether the inserted glass plates are inverted and other problems, and re-assemble the mold; if there is no leakage, the next step can be carried out.

[0054] (3) Preparation of separating gel solution: Add Tris-HCL buffer, acrylamide solution, SDS solution, APS solution, TEMED, and water to the container in sequence according to the separating gel solution formulation shown in Table 1, and mix evenly to obtain the separating gel solution.

[0055] (4) Pour out the pure water in the mold and remove the residual moisture with absorbent paper. Quickly add the prepared separating gel solution into the gap between the glass plates to the position of two-thirds of the height of the glass plates, and add ultrapure water to the remaining one-third height of the gap between the glass plates for liquid sealing for 20 min.

[0056] (5) Preparation of stacking gel solution: Add acrylamide solution, SDS solution, APS solution, TEMED, Tris-HCL buffer, and water to the container in sequence according to the stacking gel solution formulation shown in Table 1, and mix evenly to obtain the stacking gel solution.

[0057] (6) When it is observed that there is an obvious demarcation line between the separating gel layer and the water layer in the glass plates, pour out one-third of the ultrapure water in the glass plates and remove the residual moisture with absorbent paper. Quickly add the prepared stacking gel solution into the gap between the glass plates, immediately insert the comb, and let it stand for 20 min.

[0058] (7) When the stacking gel in the glass plates solidifies, remove the mold and take out the glass plates to obtain the polyacrylamide gel.

[0059] 4. Protein loading

[0060] Clamp the removed glass plate and place it in the electrophoresis tank. Remove the tooth comb, add electrophoresis solution, spot samples in the comb holes, and quantitatively load 5μg and 10μg.

[0061] Preparation method of electrophoresis buffer: weigh 3.0 g of Tris (tris(hydroxymethyl)aminomethane), 14.4 g of glycine, and 1.0 g of SDS, add appropriate amount of purified water to dilute to 1 L, and use it immediately after preparation.

[0062] 5. Electrophoresis process

[0063] After loading, adjust the voltage to 80V and run for 30 minutes. After the bromophenol blue band enters the separation gel, adjust the voltage to 120V and run for 60 minutes. Stop the electrophoresis when the bromophenol blue band moves to the bottom of the SDS-PAGE electrophoresis gel.

[0064] 6. Coomassie Brilliant Blue Staining

[0065] Take out the glass plate, pry it open carefully from the gap of the glass plate, cut off the concentrated gel, take out the separation gel, put it into the staining box, rinse with ultrapure water for 0.2 minutes, add 60mL of Coomassie Brilliant Blue staining solution, heat it on high heat in a microwave oven for 0.5 minutes, and place it on a decolorizing shaker for shaking and staining for 40 minutes.

[0066] 7. Decolorization

[0067] Pour out the staining solution, add ultrapure water and place on a decolorizing shaker for 0.2 min. Pour out the ultrapure water, add 60 mL of decolorizing solution, place on a decolorizing shaker for overnight decolorization, and then replace the decolorizing solution until the bands are clear.

[0068] 8. Take photos

[0069] Scan the gel using a gel imager and save the scan results.

[0070] Table 1 Polyacrylamide gel formula

[0071]

[0072]

[0073] Comparative Example 1 Pharmacopoeia Formula

[0074] The experiment was conducted using the method of Example 1. The difference from Example 1 is that the polyacrylamide gel was prepared using the pharmacopoeia formula. The specific formula is shown in Table 1.

[0075] Comparative Example 2 Commercial Formula

[0076] The commercial polyacrylamide gel (manufacturer: GenScript Biotech Corporation, product number: M42012C) was used to electrophoretically separate pertussis toxin PT protein under the same conditions as in Example 1.

[0077] Analysis of experimental results

[0078] The experimental results are as Figures 1 to 4 shown. Figure 1 Among them, Samples 1-3 used different batches of polyacrylamide gels prepared according to Formulation 1 of the present invention. Figure 2 Among them, Samples 4-6 used different batches of polyacrylamide gels prepared according to Formulation 2 of the present invention. Figure 1 and 2 Among them, Samples 1-6 were respectively loaded with 5 μg and 10 μg of samples in a quantitative manner. Figure 3 Among them, Sample 7 used a polyacrylamide gel prepared according to the pharmacopoeia formula. Figure 4 Among them, Sample 8 used a commercially available polyacrylamide gel.

[0079] Among them, Figure 1 and 2 clearly showed the subunit bands of S1, S2, S3, S4, and S5 of pertussis toxin PT protein. Figure 3 The subunit bands of pertussis PT protein were relatively blurred in Figure 4 and only 3 subunit bands of pertussis PT protein could be shown in , and the molecular weight shifted upward.

[0080] Compared with Figure 3 , Figure 4 , for the polyacrylamide gels prepared according to Formulation 1 and Formulation 2 of the present invention by electrophoresis, the bands of subunits S1, S2, S3, S4, and S5 of pertussis toxin PT protein were clearly visible, and had a high resolution, and could clearly separate the five subunits of pertussis toxin PT protein. Among them, Formulation 1 had a little tailing phenomenon, but it did not affect the resolution of each subunit of pertussis toxin PT protein, and Formulation 2 was better.

[0081] In addition, as Figure 1 shown, when the same batch of samples was used with different sample loading amounts, when the sample loading amount was 10 μg, there was a little tailing phenomenon in the S5 subunit, and the effect was better when the sample loading amount was 5 μg. Therefore, the preferred sample loading amount was 5 μg.

[0082] Investigation on the dosage of 1.5 mol / L Tris-HCL buffer in the separating gel solution of Example 2

[0083] The experiment was carried out according to the method of Formulation 2 in Example 1. The difference from Formulation 2 in Example 1 was that the 1.5 mol / L Tris-HCL buffer in the separating gel solution was 0.7 mL, 1.0 mL, 1.25 mL, 1.5 mL, and 1.7 mL respectively.

[0084] It was found that when 0.7 mL and 1.7 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 1.0 mL, 1.25 mL, 1.3 mL (i.e., Formulation 2 of Example 1) and 1.5 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0085] Investigation on the dosage of 30% acrylamide solution in the separating gel solution of Example 3

[0086] The experiment was carried out according to the method of Formulation 2 of Example 1. The difference from Formulation 2 of Example 1 was that the 30% acrylamide solution in the separating gel solution was 1.5 mL, 1.8 mL, 2.2 mL and 2.5 mL respectively.

[0087] It was found that when 1.5 mL and 2.5 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 1.8 mL, 2.0 mL (i.e., Formulation 2 of Example 1) and 2.2 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0088] Investigation on the dosage of 10% SDS solution in the separating gel solution of Example 4

[0089] The experiment was carried out according to the method of Formulation 2 of Example 1. The difference from Formulation 2 of Example 1 was that the 10% SDS solution in the separating gel solution was 0.02 mL, 0.03 mL, 0.08 mL and 0.09 mL respectively.

[0090] It was found that when 0.02 mL and 0.09 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 0.03 mL, 0.05 mL (i.e., Formulation 2 of Example 1) and 0.08 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0091] Investigation on the dosage of 10% APS solution in the separating gel solution of Example 5

[0092] The experiment was carried out according to the method of Formulation 2 of Example 1. The difference from Formulation 2 of Example 1 was that the 10% APS solution in the separating gel solution was 0.02 mL, 0.03 mL, 0.08 mL and 0.09 mL respectively.

[0093] It was found that when 0.02 mL and 0.09 mL were selected, the subunits S1, S2, S3, S4, and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 0.03 mL, 0.05 mL (i.e., formulation 2 of Example 1), and 0.08 mL were selected, the subunits S1, S2, S3, S4, and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0094] Investigation on the dosage of TEMED in the separating gel solution of Example 6

[0095] The experiment was carried out according to the method of formulation 2 of Example 1. The difference from formulation 2 of Example 1 was that the TEMED in the separating gel solution was 0.001 mL, 0.005 mL, 0.006 mL, and 0.007 mL respectively.

[0096] It was found that when 0.007 mL was selected, the subunits S1, S2, S3, S4, and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. When 0.001 mL, 0.002 mL (i.e., formulation 2 of Example 1), 0.005 mL, and 0.006 mL were selected, the subunits S1, S2, S3, S4, and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0097] Investigation on the dosage of water in the separating gel solution of Example 7

[0098] The experiment was carried out according to the method of formulation 2 of Example 1. The difference from formulation 2 of Example 1 was that the water in the separating gel solution was 1.5 mL, 1.7 mL, 2.2 mL, and 2.85 mL respectively.

[0099] It was found that when 2.85 mL was selected, the subunits S1, S2, S3, S4, and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. When 1.5 mL, 1.7 mL, 2.0 mL (i.e., formulation 2 of Example 1), and 2.2 mL were selected, the subunits S1, S2, S3, S4, and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0100] Investigation on the dosage of 0.5 mol / L Tris-HCL in the stacking gel solution of Example 8

[0101] The experiment was carried out according to the method of formulation 2 of Example 1. The difference from formulation 2 of Example 1 was that the 0.5 mol / L Tris-HCL in the stacking gel solution was 0.4 mL, 0.45 mL, 0.55 mL, and 0.6 mL respectively.

[0102] It was found that when 0.4 mL and 0.6 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 0.45 mL, 0.5 mL (i.e., formulation two of Example 1) and 0.55 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0103] Investigation on the dosage of 30% acrylamide in the stacking gel solution of Example 9

[0104] The experiment was carried out according to the method of formulation two of Example 1. The difference from formulation two of Example 1 was that the 30% acrylamide solution in the stacking gel solution was 0.22 mL, 0.24 mL, 0.26 mL, 0.32 mL and 0.35 mL respectively.

[0105] It was found that when 0.22 mL and 0.35 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. When 0.24 mL, 0.26 mL, 0.30 mL (i.e., formulation two of Example 1) and 0.32 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0106] Investigation on the dosage of 10% SDS solution in the stacking gel solution of Example 10

[0107] The experiment was carried out according to the method of formulation two of Example 1. The difference from formulation two of Example 1 was that the 10% SDS solution in the stacking gel solution was 0.017 mL, 0.018 mL, 0.022 mL and 0.023 mL respectively.

[0108] It was found that when 0.017 mL and 0.023 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 0.018 mL, 0.020 mL (i.e., formulation two of Example 1) and 0.022 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0109] Investigation on the dosage of 10% APS solution in the stacking gel solution of Example 11

[0110] The experiment was carried out according to the method of formulation two of Example 1. The difference from formulation two of Example 1 was that the 10% APS solution in the stacking gel solution was 0.017 mL, 0.018 mL, 0.022 mL and 0.023 mL respectively.

[0111] It was found that when 0.017 mL and 0.023 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 0.018 mL, 0.020 mL (i.e., formulation two of Example 1) and 0.022 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0112] Investigation on the dosage of TEMED in the stacking gel solution of Example 12

[0113] The experiment was carried out according to the method of formulation two of Example 1. The difference from formulation two of Example 1 was that the dosages of TEMED in the stacking gel solution were 0.0017 mL, 0.0018 mL, 0.0022 mL and 0.0023 mL respectively.

[0114] It was found that when 0.0017 mL and 0.0023 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 0.0018 mL, 0.0020 mL (i.e., formulation two of Example 1) and 0.0022 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0115] Investigation on the dosage of water in the stacking gel solution of Example 13

[0116] The experiment was carried out according to the method of formulation two of Example 1. The difference from formulation two of Example 1 was that the dosages of water in the stacking gel solution were 0.85 mL, 1.00 mL, 1.22 mL, 1.40 mL and 1.55 mL respectively.

[0117] It was found that when 0.85 mL and 1.55 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could not be shown separately, and the resolution was poor. However, when 1.00 mL, 1.16 mL (i.e., formulation two of Example 1), 1.22 mL and 1.40 mL were selected, the subunits S1, S2, S3, S4 and S5 bands of pertussis toxin PT protein could be clearly shown separately, and the resolution was good.

[0118] The above are only several exemplary embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention is disclosed above with preferred embodiments, it is not intended to limit the present invention. Any equivalent or equivalent embodiments obtained by making some changes or modifications using the disclosed technical content by those skilled in the art without departing from the technical solution of the present invention fall within the scope of the present invention.

Claims

1. A composition for preparing polyacrylamide gel, which comprises a separating gel solution and a stacking gel solution. The separating gel solution comprises: by volume parts, 1 - 1.5 parts of 1.2 - 1.8 mol / L Tris-HCL buffer, 1.8 - 2.2 parts of 20 - 40 wt% acrylamide solution, 0.03 - 0.08 parts of 5 - 15 wt% sodium dodecyl sulfate solution, 0.03 - 0.08 parts of 5 - 15 wt% ammonium persulfate solution, 0.001 - 0.006 parts of tetramethylethylenediamine, and 1.5 - 2.2 parts of water; The stacking gel solution comprises: by volume parts, 0.24 - 0.32 parts of 20 - 40 wt% acrylamide solution, 0.018 - 0.022 parts of 5 - 15 wt% sodium dodecyl sulfate solution, 0.018 - 0.022 parts of 5 - 15 wt% ammonium persulfate solution, 0.0018 - 0.0022 parts of tetramethylethylenediamine, 0.45 - 0.55 parts of 0.2 - 0.8 mol / L Tris-HCL buffer, and 1.0 - 1.4 parts of water.

2. The composition according to claim 1, wherein The separating gel solution comprises: by volume parts, 1.2 - 1.3 parts of 1.5 mol / L Tris-HCL buffer, 1.9 - 2.1 parts of 30 wt% acrylamide solution, 0.04 - 0.06 parts of 10 wt% sodium dodecyl sulfate solution, 0.04 - 0.06 parts of 10 wt% ammonium persulfate solution, 0.002 - 0.005 parts of tetramethylethylenediamine, and 1.7 - 2 parts of water; Preferably, the stacking gel solution comprises: by volume parts, 0.26 - 0.3 parts of 30 wt% acrylamide solution, 0.019 - 0.021 parts of 10 wt% sodium dodecyl sulfate solution, 0.019 - 0.021 parts of 10 wt% ammonium persulfate solution, 0.0019 - 0.0021 parts of tetramethylethylenediamine, 0.48 - 0.52 parts of 0.5 mol / L Tris-HCL buffer, and 1.1 - 1.3 parts of water.

3. The composition according to claim 1 or 2, wherein The total concentration of solutes in the separating gel solution is 10 - 15 wt%, preferably 11 - 12 wt%; Preferably, the total concentration of solutes in the stacking gel solution is 3 - 5 wt%, preferably 3.9 - 4.6 wt%.

4. The composition according to any one of claims 1 to 3, wherein, The volume ratio of the separating gel solution to the stacking gel solution in the composition is 1 - 4:1, preferably 2 - 3:

1.

5. A polyacrylamide gel, which is prepared from the composition according to any one of claims 1 to 4.

6. A method for preparing a polyacrylamide gel from the composition according to any one of claims 1 to 4, the method comprising the following steps: (1) Sequentially add the Tris-HCL buffer, acrylamide solution, sodium dodecyl sulfate solution, ammonium persulfate solution, tetramethylethylenediamine, and water in the formula amount of the separating gel solution into a container, mix evenly to obtain a separating gel solution, add the separating gel solution into a gel plate, add ultrapure water for liquid sealing, let stand, and remove the ultrapure water after the separating gel solution solidifies to obtain a separating gel layer; (2) Add acrylamide solution, sodium dodecyl sulfate solution, ammonium persulfate solution, tetramethylethylenediamine, Tris-HCL buffer solution and water in the container in sequence according to the formula amount of the stacking gel solution, mix evenly to obtain the stacking gel solution, add the stacking gel solution to the upper layer of the separating gel layer in the gel plate, insert the comb, and let it stand still. After the stacking gel solution solidifies, a polyacrylamide gel is obtained.

7. The method according to claim 6, wherein, The method further comprises the following step: before step (1), check the leak of the gel plate. Preferably, in step (1), let it stand still for 10 - 30 minutes, preferably 20 minutes. Preferably, in step (2), let it stand still for 10 - 30 minutes, preferably 20 minutes.

8. A polyacrylamide gel kit, which comprises the composition according to any one of claims 1 to 4.

9. A method for detecting polyacrylamide gel electrophoresis, wherein the polyacrylamide gel is prepared from the composition according to any one of claims 1 to 4 or from the kit according to claim 8, and the method comprises the following steps: (1) Load the protein sample into the gel wells of the polyacrylamide gel and perform electrophoresis. (2) After the electrophoresis is completed, stain and decolorize the polyacrylamide gel in sequence until the reaction bands are clear, and scan and save the reaction results. Preferably, in step (1), the loading amount of the protein sample is 5 - 10 μg, preferably 5 μg.

10. The detection method according to claim 9, wherein, The detection method is used for detecting the molecular weight of proteins. Preferably, the protein is selected from one or more of pertussis toxin, pertussis adhesin and filamentous hemagglutinin.

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