Gradient glue and its preparation method and application

Abstract

The present application relates to gradient gel and its preparation method and application, the gradient gel includes separation gel of different concentrations, and the separation gel includes components of the following concentrations: 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 1wt%-15wt% trehalose. The gradient gel has the advantages of high resolution, clear bands, good separation effect and low toxicity. The formula of the present application is more suitable for the preparation of gradient gel, and the combination of components in the formula makes it more suitable for automated glue filling equipment to realize automated production of gradient gel.

Description

Technical Field

[0001] The present application relates to the field of biochemical technology, and in particular to a gradient gel and a preparation method and application thereof. Background Art

[0002] Polyacrylamide gel electrophoresis (PAGE) is the most commonly used experimental technique for protein analysis in biological experiments. At present, traditional PAGE mainly includes polyacrylamide (acrylamide: methylene bisacrylamide = 29:1), Tris hydrochloric acid buffer, tetramethylethylenediamine and ammonium persulfate. Polyacrylamide is polymerized with ammonium persulfate (APS) as a catalyst and tetramethylethylenediamine (TEMED) as an accelerator. During the polymerization process, TEMED catalyzes ammonium persulfate to produce free radicals, which initiate the polymerization of acrylamide monomers. At the same time, methylene bisacrylamide and acrylamide chains form methylene bonds to form a three-dimensional network structure and make a gel. TEMED is a nerve agent with a foul odor. It is of great significance to reduce or eliminate the use of toxic reagents in polyacrylamide gels.

[0003] Currently, commonly used protein gels are still made manually, which has problems such as poor reproducibility and susceptibility to toxic reagents.

[0004] In conventional experiments, in order to obtain better experimental results, researchers need to make protein gels of different concentrations according to the molecular weight of the protein, which increases the workload. Gradient gels, however, can adapt to proteins of different molecular weights because they have multiple concentrations. The current gradient gels are mainly derived from commercial products, which are relatively expensive and the separation effect is not good enough. The existing formula is difficult to achieve stable production of gradient gels, and is even less suitable for automated production of gradient gels. Summary of the invention

[0005] Based on this, it is necessary for one embodiment of the present application to provide a gradient glue and a preparation method thereof, wherein the gradient glue has a good separation effect and its formula can realize stable and automated production of gradient glue.

[0006] The specific technical solutions are as follows:

[0007] In the first aspect, the present application provides a gradient gel, which comprises separation gels of different concentrations, and the separation gel comprises components of the following concentrations: 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 1wt%-15wt% trehalose.

[0008] In one embodiment, the gradient gel comprises a first separation gel, a second separation gel and a third separation gel, wherein the first separation gel comprises 12wt%-20wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 10wt%-15wt% trehalose; the second separation gel comprises 8wt%-12wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 5wt%-8wt% trehalose; the third separation gel includes 4wt%-8wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 1wt%-3wt% trehalose.

[0009] In one embodiment, the concentration of polyacrylamide in the first separation gel>the concentration of polyacrylamide in the second separation gel>the concentration of polyacrylamide in the third separation gel.

[0010] In a second aspect, the present application provides a method for preparing the gradient gel, the method comprising the following steps:

[0011] According to the components of the first separation gel, the second separation gel and the third separation gel in the gradient gel, corresponding gel solutions are prepared; and

[0012] The first separation gel, the second separation gel and the third separation gel are sequentially poured into the gel plate, a comb is inserted, and the gel is solidified to obtain a gradient gel.

[0013] In a third aspect, the present application provides a reagent for preparing a gradient gel, the reagent comprising 4wt%-20wt% acrylamide and methylenebisacrylamide mixture, 0.25M-0.4M Tris-HCl, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 1wt%-15wt% trehalose.

[0014] In a fourth aspect, the present application provides the use of the gradient gel, the gradient gel prepared by the preparation method or the reagent in separating proteins.

[0015] Compared with the traditional technology, this application has the following beneficial effects:

[0016] The gradient gel provided by the present application, which contains components such as N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, sodium bisulfite, ammonium persulfate and trehalose, has the advantages of clear bands, good separation effect and low toxicity.

[0017] The optimized formula of this application is more suitable for the preparation of gradient glue, and the combination of components in the formula makes it more suitable for automated glue filling equipment, realizing automated production of gradient glue and creating value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the gradient glue board of this application;

[0019] Figure 2 The electrophoresis diagrams of the 4-12% gradient gel prepared in Example 1 of the present application and the commercially available product; the left diagram is the gradient gel of Example 1, and the right diagram is the commercially available product a (4-12% gradient gel);

[0020] Figure 3 The electrophoresis diagrams of the 8-12% gradient gel prepared in Example 2 of the present application and the commercially available product; the left diagram is the gradient gel of Example 2, and the right diagram is the commercially available product b (8-12% gradient gel);

[0021] Figure 4 The electrophoresis diagrams of the 8-15% gradient gel prepared in Example 3 of the present application and the commercially available product; the left diagram is the gradient gel of Example 3, and the right diagram is the commercially available product c (8-15% gradient gel);

[0022] Figure 5 The effect diagram of the 4-12% gradient gel prepared in Example 1 of the present application and the 4-12% gradient gel prepared in Comparative Example 1 after gelation; the left diagram is Example 1, and the right diagram is Comparative Example 1;

[0023] Figure 6 These are the effect diagrams of the 4-12% gradient gel prepared in Example 1 of the present application and the 4-12% gradient gel prepared in Comparative Example 2 after gelation; the left diagram is Example 1, and the right diagram is Comparative Example 2. DETAILED DESCRIPTION

[0024] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0026] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0027] "Scope" disclosed herein can be limited in the form of lower limit and upper limit, and a given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a special range. The scope limited in this way can be including end values ​​or excluding end values, and any end value can be included or not included independently, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a scope. For example, if the scope of 50-130 and 80-110 is listed for a specific parameter, it is understood that the scope of 50-110 and 80-130 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are also listed, the following scope can be all expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise specified, the numerical range "ab" represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed in this article, and "0-5" is just an abbreviation of these numerical combinations.

[0028] In this document, in the "first aspect", "second aspect", "third aspect" and "fourth aspect", the terms "first", "second", "third" and "fourth" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third" and "fourth" only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0029] Herein, wt% means mass percentage.

[0030] One embodiment of the present application provides a gradient gel, including separation gels of different concentrations, the separation gel including 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate, and 1wt%-15wt% trehalose. The gradient gel has a wider separation range and can simultaneously separate proteins of larger molecular weights. The above components are contained in its formula to achieve better separation effects; in addition, the combination of components in the formula also makes the production of gradient gels have better repeatability and stability, and is more suitable for automated gel casting, realizing automated production of gradient gels, and tetramethylethylenediamine is not used in the formula, so the toxicity is low.

[0031] In a specific example, the separation gel further includes 4 wt %-20 wt % polyacrylamide and 0.25 M-0.4 M Tris.

[0032] In a specific example, the gradient gel includes a first separation gel, a second separation gel and a third separation gel, wherein the first separation gel includes 12wt%-20wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 10wt%-15wt% trehalose; the second separation gel includes 8wt%-12wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 5wt%-8wt% trehalose; the third separation gel includes 4wt%-8wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 1wt%-3wt% trehalose.

[0033] In a specific example, the concentration of polyacrylamide in the first separation gel (wt %)>the concentration of polyacrylamide in the second separation gel (wt %)>the concentration of polyacrylamide in the third separation gel (wt %).

[0034] In a specific example, the gradient gel includes a first separation gel, a second separation gel and a third separation gel, wherein the first separation gel includes 12wt%-15wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 10wt% trehalose; the second separation gel includes 8wt%-12wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 5wt% trehalose; the third separation gel includes 4wt%-8wt% polyacrylamide, 0.375M Tris, 0.05 wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15 wt% sodium bisulfite, 0.12 wt% ammonium persulfate and 1 wt% trehalose.

[0035] In a specific example, 4% to 12% gradient gel: the first separation gel includes 12wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 10wt% trehalose; the second separation gel includes 8wt% polyacrylamide, 0.375MTris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 5wt% trehalose; the third separation gel includes 4wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 1wt% trehalose.

[0036] In a specific example, 8%-12% gradient gel: the first separation gel includes 12wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 10wt% trehalose; the second separation gel includes 10wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 5wt% trehalose; the third separation gel includes 8wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 1wt% trehalose.

[0037] In a specific example, 8%-15% gradient gel: the first separation gel includes 15wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 10wt% trehalose; the second separation gel includes 12wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 5wt% trehalose; the third separation gel includes 8wt% polyacrylamide, 0.375M Tris, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 1wt% trehalose.

[0038] In one specific example, polyacrylamide is prepared from raw materials including acrylamide and methylenebisacrylamide.

[0039] In a specific example, the mass ratio of acrylamide to methylene bisacrylamide is (28-29):(1-2). Optionally, the mass ratio of acrylamide to methylene bisacrylamide is 29:1.

[0040] An embodiment of the present application provides a method for preparing the above-mentioned gradient gel, the method comprising the following steps:

[0041] Step a, preparing corresponding gel solutions according to the formulas of the first separation gel, the second separation gel and the third separation gel in the above gradient gel.

[0042] Step b, pouring the first separation gel, the second separation gel and the third separation gel into the gel plate in sequence, inserting a comb, and waiting for the gel to solidify to obtain a gradient gel.

[0043] In a specific example, automatic glue pouring equipment is used for glue pouring.

[0044] Specifically, the glue solutions of the first separation glue, the second separation glue and the third separation glue are respectively connected to the three pumps in the automated glue pouring equipment. Optionally, the glue solution of the first separation glue is connected to pump No. 1 of the automated glue pouring equipment; the glue solution of the second separation glue is connected to pump No. 2 of the automated glue pouring equipment; and the glue solution of the third separation glue is connected to pump No. 3 of the automated glue pouring equipment.

[0045] Specifically, the first separation gel solution is pumped into the gel plate first, and then the second separation gel solution is pumped into the gel plate. After the second separation gel solution is pumped into the gel plate, the third separation gel solution is pumped into the gel plate. After the gel filling is completed, a comb is inserted and waited for solidification.

[0046] In a specific example, the volume ratio of the first separation gel, the second separation gel and the third separation gel is (2-3): (2-3): (5-6). Optionally, the volume ratio of the first separation gel, the second separation gel and the third separation gel is 3:2:5.

[0047] One embodiment of the present application provides a reagent for preparing a gradient gel, which includes 4wt%-20wt% of a mixture of acrylamide and methylenebisacrylamide, 0.25M-0.4M Tris-HCl, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 1wt%-15wt% trehalose.

[0048] In a specific example, the reagent includes subpackaged reagent 1, reagent 2, and reagent 3, wherein:

[0049] Reagent 1 includes 12wt%-20wt% acrylamide and methylenebisacrylamide mixture, 0.25M-0.4MTris-HCl, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 10wt%-15wt% trehalose;

[0050] Reagent 2 includes 8wt%-12wt% acrylamide and methylenebisacrylamide mixture, 0.25M-0.4MTris-HCl, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 5wt%-8wt% trehalose;

[0051] Reagent 3 includes 4wt%-8wt% acrylamide and methylenebisacrylamide mixture, 0.25M-0.4MTris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 1wt%-3wt% trehalose.

[0052] In a specific example, reagent 1 includes 12wt%-15wt% acrylamide and methylenebisacrylamide mixture, 0.375M Tris-HCl, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 10wt% trehalose;

[0053] Reagent 2 includes 8wt%-12wt% acrylamide and methylenebisacrylamide mixture, 0.375M Tris-HCl, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 5wt% trehalose;

[0054] Reagent 3 includes 4wt%-8wt% acrylamide and methylenebisacrylamide mixture, 0.375M Tris-HCl, 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate and 1wt% trehalose.

[0055] In a specific example, the pH value of Tris-HCl is 8.0 to 9.0. Optionally, the pH value of Tris-HCl is 8.8.

[0056] One embodiment of the present application provides the use of the above-mentioned gradient gel, the gradient gel prepared by the above-mentioned preparation method or the above-mentioned reagent in separating proteins.

[0057] In the present application, the method for separating proteins includes polyacrylamide gel electrophoresis (PAGE), which has two forms, namely denaturing polyacrylamide gel electrophoresis and non-denaturing polyacrylamide gel electrophoresis.

[0058] In the non-denaturing polyacrylamide gel electrophoresis of proteins, the proteins can remain intact and are gradually separated in a gradient according to the molecular weight, shape and the amount of charge attached to the proteins. In the denaturing polyacrylamide gel electrophoresis, since protein denaturants such as SDS are added to the samples, the separation is only based on the molecular weight.

[0059] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0060] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0061] Example 1 Preparation of 4%-12% gradient glue

[0062] This embodiment provides a 4%-12% gradient glue, which includes high, medium and low concentration glues.

[0063] The concentration of high concentration gel is 12%, and the final concentration of each component is: 12wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 10wt% trehalose.

[0064] The concentration of medium concentration gel is 8%, and the final concentration of each component is: 8wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 5wt% trehalose.

[0065] The concentration of the low concentration gel is 4%, and the final concentrations of each component are: 4wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 1wt% trehalose.

[0066] Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.

[0067] The preparation method of the above 4%-12% gradient gel comprises the following steps:

[0068] (1) Configure high, medium and low concentration glue:

[0069] 12% high concentration glue: prepare the required volume of glue according to the above recipe.

[0070] 8% medium concentration glue: prepare the required volume of glue according to the above recipe.

[0071] 4% low concentration glue: prepare the required volume of glue solution according to the above recipe.

[0072] (2) Connect the high-concentration glue solution (12%) to pump 1 of the automated glue filling equipment, connect the medium-concentration glue solution (8%) to pump 2, and connect the low-concentration glue solution (4%) to pump 3.

[0073] (3) An automated program is set up, wherein pump No. 1 is started to pump 3 ml of high-concentration glue into the rubber plate (volume 6 ml); pump No. 1 is then paused, pump No. 2 is started, and 2 ml of medium-concentration glue is pumped into the rubber plate; pump No. 2 is then paused, pump No. 3 is started, and low-concentration glue is pumped into the rubber plate until the rubber plate is filled.

[0074] (4) Turn on the automatic glue filling instrument and complete the glue filling. Figure 1 As shown. Manual glue pouring can also be performed in the above manner.

[0075] After the gel was prepared, PAGE electrophoresis was performed to compare with a commercially available product of the same specification (ACE Biotechnology, catalog number: ET12412Gel). It was found that the electrophoresis bands of the gradient gel provided in this embodiment were clear, rich in details, and the overall effect was excellent. Figure 2 shown.

[0076] Example 2 Preparation of 8%-12% gradient glue

[0077] This embodiment provides an 8%-12% gradient glue, which includes high, medium and low concentration glues.

[0078] The concentration of high concentration gel is 12%, and the final concentration of each component is: 12wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 10wt% trehalose.

[0079] The concentration of medium concentration gel is 10%, and the final concentration of each component is: 10wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 5wt% trehalose.

[0080] The concentration of the low concentration gel is 8%, and the final concentrations of each component are: 8wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 1wt% trehalose.

[0081] Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.

[0082] The preparation method of the above 8%-12% gradient gel comprises the following steps:

[0083] (1) Configure high, medium and low concentration glue:

[0084] 12% high concentration glue: prepare the required volume of glue according to the above recipe.

[0085] 10% medium concentration glue: prepare the required volume of glue according to the above recipe.

[0086] 8% low concentration glue: prepare the required volume of glue according to the above recipe.

[0087] (2) Connect the high-concentration glue solution (12%) to pump 1 of the automatic glue filling equipment, connect the medium-concentration glue solution (10%) to pump 2, and connect the low-concentration glue solution (8%) to pump 3.

[0088] (3) An automated program is set up, wherein pump No. 1 is started to pump 3 ml of high-concentration glue into the rubber plate (volume 6 ml); pump No. 1 is then paused, pump No. 2 is started, and 2 ml of medium-concentration glue is pumped into the rubber plate; pump No. 2 is then paused, pump No. 3 is started, and low-concentration glue is pumped into the rubber plate until the rubber plate is filled.

[0089] (4) Turn on the automatic glue dispensing instrument to complete the glue dispensing. You can also perform manual glue dispensing in the same manner as above.

[0090] After the gel preparation was completed, PAGE electrophoresis was performed to compare with the commercially available product B (Quanshijin Biotechnology, catalog number: DG101-01-V2) of the same specification. It was found that the electrophoresis bands of the gradient gel provided in this embodiment were clear, rich in details, and the overall effect was excellent. Figure 3 shown.

[0091] Example 3 Preparation of 8%-15% gradient glue

[0092] This embodiment provides an 8%-15% gradient glue, which includes high, medium and low concentration glues.

[0093] The concentration of high concentration gel is 15%, and the final concentration of each component is: 15wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 10wt% trehalose.

[0094] The concentration of medium concentration gel is 12%, and the final concentration of each component is: 12wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 5wt% trehalose.

[0095] The concentration of the low concentration gel is 8%, and the final concentrations of each component are: 8wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, 0.12wt% ammonium persulfate, and 1wt% trehalose.

[0096] Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.

[0097] The preparation method of the above 8%-15% gradient gel comprises the following steps:

[0098] (1) Configure high, medium and low concentration glue:

[0099] 15% high concentration glue: Prepare the required volume of glue according to the above recipe.

[0100] 12% medium concentration glue: prepare the required volume of glue according to the above recipe.

[0101] 8% low concentration glue: prepare the required volume of glue according to the above recipe.

[0102] (2) Connect the high-concentration glue solution (15%) to pump 1 of the automatic glue filling equipment, connect the medium-concentration glue solution (12%) to pump 2, and connect the low-concentration glue solution (8%) to pump 3.

[0103] (3) An automated program is set up, wherein pump No. 1 is started to pump 3 ml of high-concentration glue into the rubber plate (volume 6 ml); pump No. 1 is then paused, pump No. 2 is started, and 2 ml of medium-concentration glue is pumped into the rubber plate; pump No. 2 is then paused, pump No. 3 is started, and low-concentration glue is pumped into the rubber plate until the rubber plate is filled.

[0104] (4) Turn on the automatic glue dispensing instrument to complete the glue dispensing. You can also perform manual glue dispensing in the same manner as above.

[0105] After the gel preparation was completed, PAGE electrophoresis was performed to compare with the commercially available c product of the same specification (GenScript, catalog number M00659). It was found that the electrophoresis bands of the gradient gel provided in this embodiment were clear, rich in details, and the overall effect was excellent. Figure 4 shown.

[0106] Comparative Example 1

[0107] The difference from the 4%-12% gradient glue in Example 1 is that the formulas of high, medium and low concentration glues are as follows:

[0108] The concentration of high concentration glue is 12%, and the final concentration of each component is: 12wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, and 0.12wt% ammonium persulfate.

[0109] The concentration of medium concentration gel is 8%, and the final concentration of each component is: 8wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, and 0.12wt% ammonium persulfate.

[0110] The concentration of the low concentration glue is 4%, and the final concentrations of the components are: 4wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH 8.8), 0.05wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.15wt% sodium bisulfite, and 0.12wt% ammonium persulfate.

[0111] Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.

[0112] The preparation method of the gradient gel is the same as that in Example 1.

[0113] After the gel preparation was completed, compared with the gradient gel prepared in Example 1, the interfaces between the gradient gels without trehalose provided in Comparative Example 1 were uneven, while the interfaces between the gradient gels containing trehalose provided in Example 1 were flat. Figure 5 As shown, it is explained that the gradient gel formula of the present application lacks trehalose and cannot realize the automatic preparation of gradient gel.

[0114] Comparative Example 2

[0115] The difference from the 4%-12% gradient glue in Example 1 is that the formulas of high, medium and low concentration glues are as follows:

[0116] The concentration of high concentration glue is 12%, and the final concentration of each component is: 12wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH8.8), 0.05wt% tetramethylethylenediamine, and 0.12wt% ammonium persulfate.

[0117] The concentration of the medium concentration gel is 8%, and the final concentrations of the components are: 8wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH 8.8), 0.05wt% tetramethylethylenediamine, and 0.12wt% ammonium persulfate.

[0118] The concentration of the low-concentration glue is 4%, and the final concentrations of the components are: 4wt% polyacrylamide, 0.375MTris-hydrochloric acid buffer (pH 8.8), 0.05wt% tetramethylethylenediamine, and 0.12wt% ammonium persulfate.

[0119] Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.

[0120] After the gel preparation was completed, compared with the gradient gel prepared in Example 1, the electrophoresis bands in Example 1 were clearer and more detailed, such as Figure 6 shown.

[0121] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0122] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A gradient glue, characterized in that: The gradient gel comprises a first separation gel, a second separation gel and a third separation gel, wherein: The first separation gel comprises components of the following concentrations: 12wt%-20wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate, and 10wt%-15wt% trehalose; The second separation gel comprises components of the following concentrations: 8wt%-12wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate, and 5wt%-8wt% trehalose; The third separation gel comprises components of the following concentrations: 4wt%-8wt% polyacrylamide, 0.25M-0.4M Tris, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate, and 1wt%-3wt% trehalose; Wherein, the concentration of polyacrylamide in the first separation gel>the concentration of polyacrylamide in the second separation gel>the concentration of polyacrylamide in the third separation gel; The polyacrylamide in the first separation gel, the second separation gel and the third separation gel is prepared from raw materials including acrylamide and methylene bisacrylamide.

2. The gradient adhesive according to claim 1, characterized in that: The mass ratio of acrylamide to methylene bisacrylamide is (28-29): (1-2).

3. The method for preparing the gradient glue according to claim 1 or 2, characterized in that: The method comprises the following steps: According to the components of the first separation gel, the second separation gel and the third separation gel in the gradient gel, corresponding gel solutions are prepared; and The first separation gel, the second separation gel and the third separation gel are sequentially poured into the gel plate, a comb is inserted, and the gel is solidified to obtain a gradient gel.

4. The preparation method according to claim 3, characterized in that: The volume ratio of the first separation gel, the second separation gel and the third separation gel is (2-3):(2-3):(5-6).

5. The preparation method according to claim 4, characterized in that: Use automatic glue filling equipment for glue filling.

6. A reagent for preparing the gradient gel according to claim 1 or 2, characterized in that: The reagents include reagent 1, reagent 2 and reagent 3 which are packaged separately. Reagent 1 includes 12wt%-20wt% acrylamide and methylenebisacrylamide mixture, 0.25M-0.4M Tris-HCl, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 10wt%-15wt% trehalose; Reagent 2 includes 8wt%-12wt% acrylamide and methylenebisacrylamide mixture, 0.25M-0.4M Tris-HCl, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 5wt%-8wt% trehalose; Reagent 3 includes 4wt%-8wt% acrylamide and methylenebisacrylamide mixture, 0.25M-0.4M Tris-HCl, 0.02wt%-0.1wt% N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, 0.1wt%-0.3wt% sodium bisulfite, 0.1wt%-0.2wt% ammonium persulfate and 1wt%-3wt% trehalose.

7. The reagent according to claim 6, characterized in that The pH value of the Tris-HCl is 8.0-9.

0.

8. Use of the gradient gel according to claim 1 or 2 or the reagent according to claim 6 or 7 in separating proteins.

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