Polyacrylamide gel and preparation method thereof

Abstract

The invention discloses polyacrylamide gel, the gel comprises concentrated gel and separation gel, and a gel solution of the concentrated gel comprises a polyacrylamide concentrated gel premixed solution and an initiator; a gel solution of the separation gel comprises a polyacrylamide separation gel premixed solution and an initiator; the polyacrylamide concentrated gel premix solution comprises 4 wt% of a polyacrylamide solution, a Tris-HCl buffer solution, 0.4 wt% of a coagulant and a dye; the polyacrylamide separation gel premix solution comprises 6 wt%-15 wt% of a polyacrylamide solution, a Tris-HCl buffer solution and 1.2 wt% of a coagulant; the coagulant comprises N, N, N ', N'-tetraethylethylenediamine and sodium hydrogen sulfite; the initiator comprises ammonium persulfate. By improving the components and proportion of the coagulant, the gelation time is shortened, and the harm of toxic reagent volatilization to people in the gel preparation process is reduced; meanwhile, by adding a certain proportion of halogenated acetic acid, the observation convenience and the experiment efficiency are improved.

Description

Technical Field

[0001] The invention relates to the technical field of protein electrophoresis, in particular to a polyacrylamide gel without using a tetramethylethylenediamine (TEMED) coagulant. Background Art

[0002] Gel electrophoresis is a fundamental technique in biology laboratories that can be used to separate macromolecules such as DNA, RNA, and proteins. Different separation media and mechanisms allow subsets of these molecules to be separated more efficiently by exploiting their physical properties. For proteins in particular, polyacrylamide gel electrophoresis (PAGE) is often the technique of choice. Currently, polyacrylamide is formed by polymerizing an acrylamide-bisacrylamide mixture by adding ammonium persulfate (APS), catalyzed by the coagulant tetramethylethylenediamine (TEMED), to produce a highly cross-linked gel matrix. The gel acts as a sieve through which proteins are passed under the influence of an electric field. Proteins have an overall positive or negative charge, which allows protein molecules to move toward the isoelectric point, where the molecule has no net charge. By denaturing the proteins and giving them a uniform negative charge, they can be separated based on their size as they migrate toward the positive electrode.

[0003] In actual operation, the traditional polyacrylamide gel preparation technology has the following disadvantages: (1) TEMED coagulant is a volatile, flammable, corrosive and pungent neurotropic reagent; (2) there are many types of reagents and the preparation is complicated; (3) the coagulation time is long, generally requiring waiting for 30-60 minutes; (4) traditional polyacrylamide gel is colorless and transparent, and it is not easy to see the gel holes when loading the sample, and it is easy to click the wrong gel hole, resulting in sample contamination and waste; (5) traditional polyacrylamide gel must be stained with Coomassie brilliant blue after running to observe protein bands, and the experiment time is relatively long. Summary of the invention

[0004] One of the technical problems to be solved by the present invention is to provide a polyacrylamide gel which has a short gelling time, is easy to observe and has low toxicity.

[0005] In order to solve the above technical problems, the polyacrylamide gel of the present invention comprises a concentrated gel and a separation gel, wherein the gel solution of the concentrated gel comprises a polyacrylamide concentrated gel premix and an initiator; the gel solution of the separation gel comprises a polyacrylamide separation gel premix and an initiator; the polyacrylamide concentrated gel premix comprises a 4wt% polyacrylamide solution, a Tris-HCl buffer, a 0.4wt% coagulant and a dye; the polyacrylamide separation gel premix comprises a 6wt% to 15wt% polyacrylamide solution, a Tris-HCl buffer and a 1.2wt% coagulant; the coagulant comprises N,N,N',N'-tetraethylethylenediamine and sodium bisulfite; and the initiator comprises an ammonium persulfate solution.

[0006] In the polyacrylamide concentration gel premix, the concentration of Tris-HCl buffer is 0.16 M, and the pH value is 6.8; in the polyacrylamide separation gel premix, the concentration of Tris-HCl buffer is 0.39 M, and the pH value is 8.8.

[0007] In the coagulant, the weight percentage of N,N,N',N'-tetraethylethylenediamine is 5wt% to 15wt%, and the weight percentage of sodium bisulfite is 5wt% to 16wt%.

[0008] The weight percentage concentration of the ammonium persulfate solution is preferably 0.1 wt %.

[0009] Preferably, a halogenated acetic acid with a concentration of less than 10 wt % can be added to the polyacrylamide separation gel premix; the halogenated acetic acid is preferably trichloroacetic acid or tribromoacetic acid.

[0010] The second technical problem to be solved by the present invention is to provide a kit for polyacrylamide gel, the kit comprising concentrated gel A liquid, concentrated gel B liquid, separation gel A liquid, separation gel B liquid and an initiator; wherein the concentrated gel A liquid comprises 0.16M pH 6.8 Tris-HCl buffer, 0.4wt% coagulant and dye; the concentrated gel B liquid comprises 4wt% polyacrylamide solution; the separation gel A liquid comprises 0.39M pH 8.8 Tris-HCl buffer and 1.2wt% coagulant; the separation gel B liquid comprises 6wt% to 15wt% polyacrylamide solution; the initiator comprises 0.1wt% ammonium persulfate solution; the coagulant comprises 5wt% to 15wt% N,N,N',N'-tetraethylethylenediamine and 5wt% to 16wt% sodium bisulfite.

[0011] Preferably, the separation gel A solution may further contain halogenated acetic acid at a concentration of less than 10 wt %; the halogenated acetic acid is preferably trichloroacetic acid or tribromoacetic acid.

[0012] The polyacrylamide solution is preferably a 30 wt % Acr-Bis solution, which is polymerized from acrylamide and methylene bisacrylamide in a mass ratio of 29:1.

[0013] The dye is preferably a PAGE upper layer gum red dye with a concentration of 500×, and the weight percentage concentration of the dye is preferably 0.08wt%.

[0014] The third technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned polyacrylamide gel, which comprises the following steps:

[0015] 1) preparing a polyacrylamide solution: preparing a polyacrylamide solution by polymerizing acrylamide and methylenebisacrylamide at a mass ratio of 29:1;

[0016] 2) preparing a coagulant: weighing 5wt% to 16wt% of sodium bisulfite and dissolving it in ultrapure water; taking 5wt% to 15wt% of N,N,N',N'-tetraethylethylenediamine and dissolving it in the sodium bisulfite solution, mixing well, and cooling to obtain a coagulant;

[0017] 3) preparing polyacrylamide separation gel: using ultrapure water as solvent, mixing 6wt% to 15wt% polyacrylamide solution, pH 8.8 Tris-HCl buffer and 1.2wt% coagulant to obtain a polyacrylamide separation gel premix, adding an initiator ammonium persulfate solution, mixing, injecting the gel into a gel plate, adding isopropanol to cover the surface of the gel and flattening it, and removing the alcohol layer until a clear interface appears between the interlayer gel and the alcohol layer to obtain a polyacrylamide separation gel;

[0018] 4) Preparation of polyacrylamide concentrated gel: using ultrapure water as solvent, 4 wt% polyacrylamide solution, pH 6.8 Tris-HCl buffer, 0.4 wt% coagulant and dye are mixed to obtain a polyacrylamide concentrated gel premix, and an initiator ammonium persulfate solution is added and mixed. The gel is injected onto the polyacrylamide separation gel, and a clean comb is immediately inserted and allowed to stand until the gel is polymerized to obtain the polyacrylamide concentrated gel.

[0019] In the above step 3), the concentration of Tris-HCl buffer is preferably 0.39M; in step 4), the concentration of Tris-HCl buffer is preferably 0.16M.

[0020] In the above steps 3) and 4), the weight percentage concentration of the initiator ammonium persulfate solution is preferably 0.1wt%; the dye is preferably a PAGE upper layer gum red dye with a concentration of 500×, and the weight percentage concentration is preferably 0.08wt%.

[0021] Compared with the traditional polyacrylamide gel using TEMED coagulant, the polyacrylamide gel of the present invention has the following advantages and beneficial effects:

[0022] (1) In the process of preparing polyacrylamide gel, the present invention uses a coagulant composed of N,N,N',N'-tetraethylethylenediamine and sodium bisulfite instead of the traditional volatile TEMED coagulant, thereby reducing the harm to researchers caused by the volatilization of toxic reagents;

[0023] (2) The present invention accelerates the polymerization of polyacrylamide and shortens the gel time by optimizing the ratio of N,N,N',N'-tetraethylethylenediamine and sodium bisulfite, thereby solving the problem of long gel time of traditional polyacrylamide and improving the efficiency of the experiment without affecting the protein separation effect;

[0024] (3) The present invention adds PAGE upper gel red dye to the concentrated gel of polyacrylamide gel, which makes it convenient for researchers to load samples and judge whether to change the electrophoresis conditions, so that clearer and more beautiful electrophoresis bands can be obtained;

[0025] (4) The present invention adds a certain proportion of halogenated acetic acid to the separation gel of polyacrylamide gel. The compound can bind to protein under specific ultraviolet light excitation and emit fluorescence. After the polyacrylamide gel running is completed, the ultraviolet function of the microscopic gel imaging system can be used to directly take pictures and observe the protein bands without the need for Coomassie brilliant blue staining, thereby shortening the experimental time and greatly improving the experimental efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the electrophoresis effect of the 12% polyacrylamide gel prepared in Example 1 after solidification;

[0027] Figure 2 This is a picture showing the effect of adding a protein sample to the 12% polyacrylamide gel prepared in Example 1 and directly observing it under a microscopic gel imaging system;

[0028] Figure 3 This is a picture showing the effect of electrophoresis of a protein sample added to the 12% polyacrylamide gel prepared in Example 1 and then stained with Coomassie Brilliant Blue for about 15 minutes;

[0029] Figure 4 This is a diagram showing the electrophoresis effect of the 10% polyacrylamide gel prepared in Example 2 after solidification;

[0030] Figure 5 This is a picture of the effect of adding a protein marker to the 10% polyacrylamide gel prepared in Example 2 and performing electrophoresis, and then directly observing it under a microscopic gel imaging system;

[0031] Figure 6 This is a diagram showing the electrophoresis effect of the 8% polyacrylamide gel prepared in Example 3 after solidification;

[0032] Figure 7 This is a picture showing the effect of adding protein marker to the 8% polyacrylamide gel prepared in Example 3 and directly observing it under a microscopic gel imaging system;

[0033] Figure 8 This is a picture showing the effect of adding protein marker to the 15% polyacrylamide gel prepared in Example 4 and directly observing it under a microscopic gel imaging system;

[0034] Fig. 9 This is a picture showing the effect of adding a protein sample to the 10% polyacrylamide gel prepared in Comparative Example 1 for electrophoresis and then staining with Coomassie Brilliant Blue for about 15 minutes;

[0035] Fig.10 This is a graph showing the electrophoresis effect of the 12% polyacrylamide gel prepared in Comparative Example 2 after solidification;

[0036] Fig.11 This is a diagram showing the effect of adding protein marker to the 12% polyacrylamide gel prepared in comparative example 2 after electrophoresis. DETAILED DESCRIPTION

[0037] In order to have a more specific understanding of the technical content, characteristics and effects of the present invention, the technical solution of the present invention is further described in detail in conjunction with the accompanying drawings and specific embodiments.

[0038] The protein marker used in the following examples and comparative examples is a colored pre-stained protein molecular weight standard (10-180 kDa) produced by Shanghai Baishengyue Biotechnology Co., Ltd., with the catalog number BR2001816; the protein sample is 0.5% bovine serum albumin (BSA); the Coomassie Brilliant Blue is a Coomassie Brilliant Blue Ultra-Fast Staining Solution (no decolorization required) produced by Shanghai Baishengyue Biotechnology Co., Ltd., with the catalog number BR2000087; the PAGE upper layer gum red dye is produced by MeilunBio, with the catalog number MA0441-2.

[0039] Example 1 12% polyacrylamide gel

[0040] The polyacrylamide gel of this embodiment includes a concentrated gel and a separation gel. The raw materials of the concentrated gel include 4wt% polyacrylamide, 1.5M Tris-HCl buffer (pH6.8), a non-TEMED coagulant, a PAGE upper gel red dye with a concentration of 500×, and a high-efficiency initiator; the raw materials of the separation gel include 12wt% polyacrylamide, 1.5M Tris-HCl buffer (pH8.8), a non-TEMED coagulant, a concentration of 9.5wt% trichloroacetic acid, and a high-efficiency initiator. The high-efficiency initiator is ammonium persulfate.

[0041] The polyacrylamide is prepared by polymerizing acrylamide and methylenebisacrylamide in a mass ratio of 29:1.

[0042] The method for preparing the polyacrylamide gel of this embodiment comprises the following steps:

[0043] (1) According to the proportions in Table 1, a 30 wt% Acr-Bis solution was prepared using ultrapure water as the solvent.

[0044] Table 1

[0045] Acrylamide 29wt% Methylene Bisacrylamide 1wt%

[0046] (2) According to the proportions in Table 2, use ultrapure water as the solvent to prepare a non-TEMED coagulant. The specific method is as follows: weigh 7.0 wt% of sodium bisulfite and dissolve it in ultrapure water; absorb 6.5 wt% of N,N,N',N'-tetraethylethylenediamine and dissolve it in the sodium bisulfite solution, vortex mix, and cool to obtain the non-TEMED coagulant.

[0047] Table 2

[0048] N,N,N',N'-Tetraethylethylenediamine 6.5wt% Sodium bisulfite 7.0wt%

[0049] (3) Prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 6.8; prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 8.8; prepare a 10 wt% ammonium persulfate solution. All solvents are ultrapure water.

[0050] (4) Preparation of polyacrylamide separation gel: According to the ratio in Table 3, ultrapure water is used as the solvent to prepare a 12 wt% polyacrylamide separation gel premix. After thorough mixing, take 6 mL of the 12 wt% polyacrylamide separation gel premix, add 0.1 wt% ammonium persulfate solution, mix thoroughly, and then use a pipette to quickly inject the gel into the gel plate. Then add isopropanol to cover the surface of the gel and flatten it to ensure that there are no bubbles in the gel. Wait until a clear interface appears between the interlayer gel and the alcohol layer, indicating that the gel has been polymerized (waiting time 10 min), and then remove the alcohol layer.

[0051] Table 3

[0052] Tris-HCl buffer (pH 8.8) 0.39M Non-TEMED coagulant 1.2wt% 30wt%Acr-Bis 12wt% Trichloroacetic acid 9.5wt%

[0053] (5) Prepare polyacrylamide concentrated gel: Prepare 4 wt% polyacrylamide concentrated gel premix according to the ratio in Table 4, using ultrapure water as solvent. After fully mixing, take 2 mL of 4 wt% polyacrylamide concentrated gel premix, add 0.1 wt% ammonium persulfate solution, mix fully, and then use a pipette to quickly inject the gel onto the separation gel. Immediately insert the cleaned comb and wait for the gel to polymerize (7-10 min). After polymerization, remove the comb from the electrophoresis buffer to prepare the sample well.

[0054] Table 4

[0055] Tris-HCl buffer (pH 6.8) 0.16M Non-TEMED coagulant 0.4wt% 500×PAGE upper layer gel red dye 0.08wt% 30wt%Acr-Bis 4wt%

[0056] The effect of the 12% polyacrylamide gel prepared in this example after solidification is shown in Figure 1 The red dye of PAGE upper gel was added to the upper gel to make the gel holes clearly visible and easy to load samples. The total gel preparation and gelation time was 16 min.

[0057] Figure 2The results show that the 12% polyacrylamide gel prepared in this example was added to the protein sample for electrophoresis and then directly observed under the microscopic gel imaging system. Figure 2 It can be seen that the protein bands are very clear, which is consistent with the effect obtained by staining with Coomassie Brilliant Blue for 15 minutes (see Figure 3 shown).

[0058] Example 2 10% polyacrylamide gel

[0059] The polyacrylamide gel of this embodiment includes a concentrated gel and a separation gel. The raw materials of the concentrated gel include 4wt% polyacrylamide, 1.5M Tris-HCl buffer (pH6.8), a non-TEMED coagulant, and a PAGE upper gel red dye with a concentration of 500× and a high-efficiency initiator; the raw materials of the separation gel include 10wt% polyacrylamide, 1.5M Tris-HCl buffer (pH8.8), a non-TEMED coagulant, 0.1wt% trichloroacetic acid and a high-efficiency initiator. The high-efficiency initiator is ammonium persulfate.

[0060] The polyacrylamide is prepared by polymerizing acrylamide and methylenebisacrylamide in a mass ratio of 29:1.

[0061] The method for preparing the polyacrylamide gel comprises the following steps:

[0062] (1) According to the proportions in Table 1 of Example 1, a 30 wt% Acr-Bis solution was prepared using ultrapure water as the solvent.

[0063] (2) According to the proportions in Table 5, use ultrapure water as the solvent to prepare a non-TEMED coagulant. The specific method is as follows: weigh 11.0 wt% of sodium bisulfite and dissolve it in ultrapure water; absorb 10.5 wt% of N,N,N',N'-tetraethylethylenediamine and dissolve it in the sodium bisulfite solution, vortex mix, and cool it to obtain the non-TEMED coagulant.

[0064] Table 5

[0065] N,N,N',N'-Tetraethylethylenediamine 10.5wt% Sodium bisulfite 11.0wt%

[0066] (3) Prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 6.8; prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 8.8; prepare a 10 wt% ammonium persulfate solution. All solvents are ultrapure water.

[0067] (4) Preparation of polyacrylamide separation gel: According to the ratio in Table 6, ultrapure water is used as the solvent to prepare a 10 wt% polyacrylamide separation gel premix. After thorough mixing, take 6 mL of the 10 wt% polyacrylamide separation gel premix, add 0.1 wt% ammonium persulfate solution, mix thoroughly, and then use a pipette to quickly inject the gel into the gel plate. Then add isopropanol to cover the surface of the gel and flatten it to ensure that there are no bubbles in the gel. Wait until a clear interface appears between the interlayer gel and the alcohol layer, indicating that the gel has been polymerized (waiting time 8 min), and then remove the alcohol layer.

[0068] Table 6

[0069] Tris-HCl buffer (pH 8.8) 0.39M Non-TEMED coagulant 1.2wt% 30wt%Acr-Bis 10wt% Trichloroacetic acid 0.1wt%

[0070] (5) Preparation of polyacrylamide concentrated gel: According to the ratio of Table 4 in Example 1, ultrapure water is used as the solvent to prepare a 4 wt % polyacrylamide concentrated gel premix. After thorough mixing, take 2 mL of the 4 wt % polyacrylamide concentrated gel premix, add 0.1 wt % ammonium persulfate solution, mix thoroughly, and then use a pipette to quickly inject the gel onto the separation gel. Immediately insert a clean comb and wait for the gel to polymerize (7-8 min). After polymerization, remove the comb from the electrophoresis buffer to prepare the sample loading well.

[0071] The effect of the 10% polyacrylamide gel prepared in this example after solidification can be found in Figure 4 As shown, PAGE upper gel red dye was added to the upper gel to make the gel wells clearly visible and easy to load samples. The total gel preparation and gel time was 18 minutes.

[0072] Figure 5 The results show that the 10% polyacrylamide gel prepared in this example was directly observed under a microscopic gel imaging system after adding a protein marker for electrophoresis. Figure 5 It can be seen that the protein bands are clear.

[0073] Example 3 8% polyacrylamide gel

[0074] The polyacrylamide gel of this embodiment includes a concentrated gel and a separation gel. The raw materials of the concentrated gel include 4wt% polyacrylamide, 1.5M Tris-HCl buffer (pH6.8), a non-TEMED coagulant, and a PAGE upper gel red dye with a concentration of 500× and a high-efficiency initiator; the raw materials of the separation gel include 8wt% polyacrylamide, 1.5M Tris-HCl buffer (pH8.8), a non-TEMED coagulant, a concentration of 4.6wt% trichloroacetic acid and a high-efficiency initiator. The high-efficiency initiator is ammonium persulfate.

[0075] The polyacrylamide is prepared by polymerizing acrylamide and methylenebisacrylamide in a mass ratio of 29:1.

[0076] The method for preparing the polyacrylamide gel comprises the following steps:

[0077] (1) According to the proportions in Table 1 of Example 1, a 30 wt% Acr-Bis solution was prepared using ultrapure water as the solvent.

[0078] (2) According to the proportions in Table 7, use ultrapure water as the solvent to prepare a non-TEMED coagulant. The specific method is as follows: weigh 10.0 wt% of sodium bisulfite and dissolve it in ultrapure water; absorb 7.4 wt% of N,N,N',N'-tetraethylethylenediamine and dissolve it in the sodium bisulfite solution, vortex mix, and cool to obtain the non-TEMED coagulant.

[0079] Table 7

[0080] N,N,N',N'-Tetraethylethylenediamine 7.4wt% Sodium bisulfite 10.0wt%

[0081] (3) Prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 6.8; prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 8.8; prepare a 10 wt% ammonium persulfate solution. All solvents are ultrapure water.

[0082] (4) Preparation of polyacrylamide separation gel: According to the ratio in Table 8, ultrapure water is used as the solvent to prepare 8 wt% polyacrylamide separation gel premix. After thorough mixing, take 6 mL of 8 wt% polyacrylamide separation gel premix, add 0.1 wt% ammonium persulfate solution, mix thoroughly, and then use a pipette to quickly inject the gel into the gel plate. Then add isopropanol to cover the surface of the gel and flatten it to ensure that there are no bubbles in the gel. Wait until a clear interface appears between the interlayer gel and the alcohol layer, indicating that the gel has been polymerized (waiting time 8 min), and then remove the alcohol layer.

[0083] Table 8

[0084] Tris-HCl buffer (pH 8.8) 0.39M Non-TEMED coagulant 1.2wt% 30wt%Acr-Bis 8wt% Trichloroacetic acid 4.6wt%

[0085] (5) Preparation of polyacrylamide concentrated gel: According to the ratio of Table 4 in Example 1, ultrapure water is used as the solvent to prepare a 4 wt % polyacrylamide concentrated gel premix. After thorough mixing, take 2 mL of the 4 wt % polyacrylamide concentrated gel premix, add 0.1 wt % ammonium persulfate solution, mix thoroughly, and then use a pipette to quickly inject the gel onto the separation gel. Immediately insert a clean comb and wait for the gel to polymerize (7-8 min). After polymerization, remove the comb from the electrophoresis buffer to prepare the sample loading well.

[0086] The effect of the 8% polyacrylamide gel prepared in this example after solidification is shown in Figure 6As shown, PAGE red dye was added to the upper gel to make the gel wells clearly visible and easy to load. The total gel preparation and gel time was 19 minutes.

[0087] Figure 7 The results show that the 8% polyacrylamide gel prepared in this example was added with protein marker and then directly observed under the microscopic gel imaging system. Figure 7 It can be seen that the protein bands are clear.

[0088] Example 4 15% polyacrylamide gel

[0089] The polyacrylamide gel of this embodiment includes a concentrated gel and a separation gel. The raw materials of the concentrated gel include 4wt% polyacrylamide, 1.5M Tris-HCl buffer (pH6.8), a non-TEMED coagulant, and a PAGE upper gel red dye with a concentration of 500× and a high-efficiency initiator; the raw materials of the separation gel include 15wt% polyacrylamide, 1.5M Tris-HCl buffer (pH8.8), a non-TEMED coagulant, 0.3wt% tribromoacetic acid and a high-efficiency initiator. The high-efficiency initiator is ammonium persulfate.

[0090] The polyacrylamide is prepared by polymerization of acrylamide and methylenebisacrylamide at a mass ratio of 29:1.

[0091] The method for preparing the polyacrylamide gel comprises the following steps:

[0092] (1) According to the proportions in Table 1 of Example 1, a 30 wt% Acr-Bis solution was prepared using ultrapure water as the solvent.

[0093] (2) According to the proportions in Table 9, use ultrapure water as the solvent to prepare a non-TEMED coagulant. The specific method is as follows: weigh 15.2 wt% of sodium bisulfite and dissolve it in ultrapure water; absorb 3.2 wt% of N,N,N',N'-tetraethylethylenediamine and dissolve it in the sodium bisulfite solution, vortex mix, and cool to obtain the non-TEMED coagulant.

[0094] Table 9

[0095] N,N,N',N'-Tetraethylethylenediamine 3.2wt% Sodium bisulfite 15.2wt%

[0096] (3) Prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 6.8; prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 8.8; prepare a 10 wt% ammonium persulfate solution. All solvents are ultrapure water.

[0097] (4) Preparation of polyacrylamide separation gel: According to the ratio in Table 10, use ultrapure water as the solvent to prepare a 15 wt% polyacrylamide separation gel premix. After fully mixing, take 6 mL of the 15 wt% polyacrylamide separation gel premix, add 0.1 wt% ammonium persulfate solution, mix thoroughly, and then use a pipette to quickly inject the gel into the gel plate. Then add isopropanol to cover the surface of the gel and flatten it to ensure that there are no bubbles in the gel. Wait until a clear interface appears between the interlayer gel and the alcohol layer, indicating that the gel has been polymerized (waiting time 8 min), and then remove the alcohol layer.

[0098] Table 10

[0099] Tris-HCl buffer (pH 8.8) 0.39M Non-TEMED coagulant 1.2wt% 30wt%Acr-Bis 15wt% Tribromoacetic acid 0.3wt%

[0100] (5) Preparation of polyacrylamide concentrated gel: According to the ratio of Table 4 in Example 1, ultrapure water is used as the solvent to prepare a 4 wt % polyacrylamide concentrated gel premix. After thorough mixing, take 2 mL of the 4 wt % polyacrylamide concentrated gel premix, add 0.1 wt % ammonium persulfate solution, mix thoroughly, and then use a pipette to quickly inject the gel onto the separation gel. Immediately insert a clean comb and wait for the gel to polymerize (7-8 min). After polymerization, remove the comb from the electrophoresis buffer to prepare the sample loading well.

[0101] Figure 8 The results show that the 15% polyacrylamide gel prepared in this example was directly observed under a microscopic gel imaging system after adding a protein marker for electrophoresis. Figure 8 It can be seen that the protein bands are clear.

[0102] Comparative Example 1 10% polyacrylamide gel

[0103] The polyacrylamide gel of this comparative example comprises a concentrated gel and a separation gel. The raw materials of the concentrated gel comprise 4wt% polyacrylamide, 1.5M Tris-HCl buffer (pH6.8), N,N,N',N'-tetraethylethylenediamine, sodium bisulfite, 500× PAGE upper gel red dye and a high-efficiency initiator; the raw materials of the separation gel comprise 10wt% polyacrylamide, 1.5M Tris-HCl buffer (pH8.8), N,N,N',N'-tetraethylethylenediamine, sodium bisulfite and a high-efficiency initiator. The high-efficiency initiator is ammonium persulfate.

[0104] The polyacrylamide is prepared by polymerization of acrylamide and methylenebisacrylamide at a mass ratio of 29:1.

[0105] The method for preparing the polyacrylamide gel comprises the following steps:

[0106] (1) According to the proportions in Table 1 of Example 1, a 30 wt% Acr-Bis solution was prepared using ultrapure water as the solvent.

[0107] (2) Prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 6.8; prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 8.8; prepare a 10 wt% ammonium persulfate solution. All solvents are ultrapure water.

[0108] (3) Prepare polyacrylamide separation gel: Prepare 10 wt% polyacrylamide separation gel premix according to the ratio in Table 11, using ultrapure water as solvent, and mix well. Take 6 mL of 10 wt% polyacrylamide separation gel premix, add ammonium persulfate solution with a final concentration of 0.1 wt%, mix well, and then use a pipette to quickly inject the gel into the gel plate, then add isopropanol to cover the surface of the gel and flatten it, ensuring that there are no bubbles in the gel, wait until a clear interface appears between the interlayer gel and the alcohol layer, indicating that the gel has been polymerized (waiting time is about 20-30 min), and remove the alcohol layer.

[0109] Table 11

[0110] Tris-HCl buffer (pH 8.8) 0.39M 30wt%Acr-Bis 10wt% N,N,N',N'-Tetraethylethylenediamine 0.2wt% Sodium bisulfite 1.5wt%

[0111] (4) Prepare polyacrylamide concentrated gel: Prepare 4 wt% polyacrylamide concentrated gel premix according to the proportions in Table 12, using ultrapure water as the solvent, and mix well. Take 2 mL of 4 wt% polyacrylamide concentrated gel premix, add ammonium persulfate solution with a final concentration of 0.1 wt%, mix well, and then quickly inject the gel onto the separation gel with a pipette, immediately insert the cleaned comb, and wait for the gel to polymerize (20-30 min). After polymerization, remove the comb from the electrophoresis buffer to prepare the sample well.

[0112] Table 12

[0113] Tris-HCl buffer (pH 6.8) 0.16M 500×PAGE upper layer gel red dye 0.08wt% 30wt%Acr-Bis 4wt% N,N,N',N'-Tetraethylethylenediamine 0.16wt% Sodium bisulfite 0.75wt%

[0114] (5) Coomassie Brilliant Blue Staining

[0115] After electrophoresis, rinse the polyacrylamide gel in pure water for several minutes to remove impurities and reduce the background. Discard the pure water, add an appropriate amount of ultra-fast Coomassie Brilliant Blue staining solution to cover the gel, and stain on a horizontal shaker at room temperature for 10 to 15 minutes. After staining, discard the staining solution, add pure water to wash to remove the residual staining solution, and observe the results.

[0116] When preparing polyacrylamide gel in Comparative Example 1, it takes about 40 min to 60 min from gel preparation to solidification. Fig. 9 The results are shown in Figure 1. The 10% polyacrylamide gel prepared in this comparative example was added with a protein marker for electrophoresis and then stained with Coomassie brilliant blue for observation.

[0117] The coagulation time of the polyacrylamide gel prepared by the method of Examples 1-4 is shorter, and since the step of Coomassie Brilliant Blue staining is omitted, the reagent cost is saved and the experimental efficiency is further improved.

[0118] Comparative Example 2 12% polyacrylamide gel

[0119] The polyacrylamide gel of this comparative example comprises a concentrated gel and a separation gel. The raw materials of the concentrated gel comprise 4wt% polyacrylamide, 1.5M Tris-HCl buffer (pH6.8), a non-TEMED coagulant, a PAGE upper gel red dye with a concentration of 500×, and a high-efficiency initiator; the raw materials of the separation gel comprise 12wt% polyacrylamide, 1.5M Tris-HCl buffer (pH8.8), a non-TEMED coagulant, and a high-efficiency initiator; and the high-efficiency initiator is ammonium persulfate.

[0120] The polyacrylamide is prepared by polymerization of acrylamide and methylenebisacrylamide at a mass ratio of 29:1.

[0121] The preparation method of the polyacrylamide gel of this comparative example comprises the following steps:

[0122] (1) According to the proportions in Table 1 of Example 1, a 30 wt% Acr-Bis solution was prepared using ultrapure water as the solvent.

[0123] (2) According to the proportions in Table 5 of Example 2, a non-TEMED coagulant was prepared using ultrapure water as a solvent. The preparation method was the same as that of Example 2.

[0124] (3) Prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 6.8; prepare a Tris-HCl buffer solution with a concentration of 1.5 M and a pH of 8.8; prepare a 12 wt% ammonium persulfate solution. All solvents are ultrapure water.

[0125] (4) Preparation of polyacrylamide separation gel: According to the ratio in Table 11, use ultrapure water as the solvent to prepare a 12 wt% polyacrylamide separation gel premix. After fully mixing, take 6 mL of the 12 wt% polyacrylamide separation gel premix, add a final concentration of 0.1 wt% ammonium persulfate solution, mix thoroughly, and then use a pipette to quickly inject the gel into the gel plate. Then add isopropanol to cover the surface of the gel and flatten it to ensure that there are no bubbles in the gel. Wait until a clear interface appears between the interlayer gel and the alcohol layer, indicating that the gel has been polymerized (waiting time is about 6 min), and then remove the alcohol layer.

[0126] Table 11

[0127] Tris-HCl buffer (pH 8.8) 0.39M Non-TEMED coagulant 4wt% 30wt%Acr-Bis 12wt%

[0128] (5) Prepare polyacrylamide concentrated gel: Prepare 4 wt% polyacrylamide concentrated gel premix according to the ratio in Table 12, using ultrapure water as solvent. After fully mixing, take 2 mL of 4 wt% polyacrylamide concentrated gel premix, add ammonium persulfate solution with a final concentration of 0.1 wt%, and then use a pipette to quickly inject the gel onto the separation gel. Immediately insert the cleaned comb and wait for the gel to polymerize (3-5 min). After polymerization, remove the comb from the electrophoresis buffer before use to prepare the sample loading well.

[0129] Table 12

[0130] Tris-HCl buffer (pH 6.8) 0.16M Non-TEMED coagulant 1.7wt% 500×PAGE upper layer gel red dye 0.08wt% 30wt%Acr-Bis 4wt%

[0131] (5) Coomassie Brilliant Blue Staining

[0132] After electrophoresis, rinse the polyacrylamide gel in pure water for several minutes to remove impurities and reduce the background. Discard the pure water, add an appropriate amount of ultra-fast Coomassie Brilliant Blue staining solution to cover the gel, and stain on a horizontal shaker at room temperature for 10 to 15 minutes. After staining, discard the staining solution, add pure water to wash and remove the residual staining solution, and then observe the results.

[0133] The effect of the 12% polyacrylamide gel prepared in this comparative example 2 after solidification is as follows Fig.10 As shown in the figure, the glue hole is curved and there is a lot of residual glue. The total time for glue preparation and gelation is about 10 minutes.

[0134] Fig.11 The results show the effect of adding protein marker to the 12% polyacrylamide gel prepared in Comparative Example 2 for electrophoresis.

[0135] When preparing polyacrylamide gel in comparative example 2, it takes about 10 minutes from gel preparation to gel coagulation. However, due to the large amount of coagulant used, the gel time is too fast, which makes the gel coagulation uneven, resulting in the gel holes being crooked after the upper gel comb is pulled out, a lot of residual gel, and the sample is easy to overflow to other holes, and the bands will become blurred during the gel running process.

[0136] The method of Examples 1-4 is used to prepare polyacrylamide gel, which not only takes into account the gel preparation and gel time, but also ensures that there is basically no residual gel in the upper layer of gel, and there will be no bending of gel holes, overflow of samples, etc.

[0137] The above embodiments are merely feasible or preferred embodiments of the present invention and are used to illustrate the present invention rather than to limit the scope of the patent application of the present invention. Therefore, all equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope covered by the patent of the present invention.

Claims

1. A polyacrylamide gel, comprising a concentration gel and a separation gel, characterized in that: The gel solution of the concentrated gel comprises a polyacrylamide concentrated gel premix and an initiator; the gel solution of the separation gel comprises a polyacrylamide separation gel premix and an initiator; the polyacrylamide concentrated gel premix comprises a 4wt% polyacrylamide solution, a Tris-HCl buffer, a 0.4wt% coagulant and a dye; the polyacrylamide separation gel premix comprises a 6wt% to 15wt% polyacrylamide solution, a Tris-HCl buffer and a 1.2wt% coagulant; the coagulant comprises N,N,N',N'-tetraethylethylenediamine and sodium bisulfite; the initiator comprises an ammonium persulfate solution.

2. The polyacrylamide gel according to claim 1, characterized in that The polyacrylamide solution is a 30wt% Acr-Bis solution, which is polymerized from acrylamide and methylenebisacrylamide in a mass ratio of 29:

1.

3. The polyacrylamide gel according to claim 1, characterized in that In the polyacrylamide concentration gel premix, the concentration of Tris-HCl buffer is 0.16 M, and the pH value is 6.8; in the polyacrylamide separation gel premix, the concentration of Tris-HCl buffer is 0.39 M, and the pH value is 8.

8.

4. The polyacrylamide gel according to claim 1, characterized in that In the coagulant, the weight percentage of N,N,N',N'-tetraethylethylenediamine is 5wt% to 15wt%, and the weight percentage of sodium bisulfite is 5wt% to 16wt%.

5. The polyacrylamide gel according to claim 1, characterized in that The weight percentage concentration of the ammonium persulfate solution is 0.1 wt %.

6. The polyacrylamide gel according to claim 1, characterized in that The polyacrylamide separation gel premix also contains less than 10 wt % of halogenated acetic acid.

7. The polyacrylamide gel according to claim 6, characterized in that The halogenated acetic acid is trichloroacetic acid or tribromoacetic acid.

8. The polyacrylamide gel according to claim 1, characterized in that The dye is a PAGE upper layer gum red dye with a concentration of 500×, and the weight percentage concentration of the dye is 0.08wt%.

9. The method for preparing the polyacrylamide gel according to any one of claims 1 to 8, characterized in that: The steps include: 1) preparing a polyacrylamide solution: preparing a polyacrylamide solution by polymerizing acrylamide and methylenebisacrylamide at a mass ratio of 29:1; 2) preparing a coagulant: weighing 5wt% to 16wt% of sodium bisulfite and dissolving it in ultrapure water; taking 5wt% to 15wt% of N,N,N',N'-tetraethylethylenediamine and dissolving it in the sodium bisulfite solution, mixing well, and cooling to obtain a coagulant; 3) preparing polyacrylamide separation gel: using ultrapure water as solvent, mixing 6wt% to 15wt% polyacrylamide solution, pH 8.8 Tris-HCl buffer and 1.2wt% coagulant to obtain a polyacrylamide separation gel premix, adding an initiator ammonium persulfate solution, mixing, injecting the gel into a gel plate, adding isopropanol to cover the surface of the gel and flattening it, and removing the alcohol layer until a clear interface appears between the interlayer gel and the alcohol layer to obtain a polyacrylamide separation gel; 4) Preparation of polyacrylamide concentrated gel: using ultrapure water as solvent, 4 wt% polyacrylamide solution, pH 6.8 Tris-HCl buffer, 0.4 wt% coagulant and dye are mixed to obtain a polyacrylamide concentrated gel premix, and an initiator ammonium persulfate solution is added and mixed. The gel is injected onto the polyacrylamide separation gel, and a clean comb is immediately inserted and allowed to stand until the gel is polymerized to obtain the polyacrylamide concentrated gel.

10. The method according to claim 9, characterized in that Step 3), the concentration of Tris-HCl buffer is 0.39M; step 4), the concentration of Tris-HCl buffer is 0.16M; the weight percentage concentration of the initiator ammonium persulfate solution is 0.1wt%; the dye is a PAGE upper layer gum red dye with a concentration of 500× and a weight percentage concentration of 0.08wt%.

11. A kit for polyacrylamide gel, characterized in that: The kit comprises concentrated gel A liquid, concentrated gel B liquid, separation gel A liquid, separation gel B liquid and an initiator; wherein the concentrated gel A liquid comprises 0.16M Tris-HCl buffer with pH 6.8, 0.4wt% coagulant and dye; the concentrated gel B liquid comprises 4wt% polyacrylamide solution; the separation gel A liquid comprises 0.39M Tris-HCl buffer with pH 8.8 and 1.2wt% coagulant; the separation gel B liquid comprises 6wt% to 15wt% polyacrylamide solution; the initiator comprises 0.1wt% ammonium persulfate solution; and the coagulant comprises 5wt% to 15wt% N,N,N',N'-tetraethylethylenediamine and 5wt% to 16wt% sodium bisulfite.

12. The polyacrylamide gel according to claim 11, characterized in that The polyacrylamide solution is a 30wt% Acr-Bis solution, which is polymerized from acrylamide and methylenebisacrylamide in a mass ratio of 29:

1.

13. The polyacrylamide gel according to claim 11, characterized in that The separation gel A solution also contains less than 10 wt % of halogenated acetic acid.

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