Sds-page gels containing hydroxymethylcellulose and methods for their preparation

Abstract

This invention relates to the field of biochemistry and molecular biology experimental technology, specifically to an SDS-PAGE gel containing hydroxymethyl cellulose, its preparation method, and its applications. The SDS-PAGE gel is polymerized from acrylamide monomer, N,N'-methylenebisacrylamide, sodium dodecyl sulfate, buffer reagent, initiator system, and reinforcing agent. This gel significantly improves mechanical strength, toughness, and tear resistance while maintaining excellent electrophoretic resolution and transparency, effectively solving the problems of traditional gels being fragile, difficult to handle, and easily damaged during transfer processes. The preparation method of this invention is simple and low-cost, and suitable for biochemical research fields such as protein electrophoresis analysis and Western blotting.

Description

Technical Field

[0001] This invention belongs to the field of experimental technology of biochemistry and molecular biology, and specifically relates to a polyacrylamide gel electrophoresis material for protein separation and analysis, particularly an SDS-PAGE gel with added hydroxymethyl cellulose to enhance its mechanical strength and toughness, and its preparation method. Background Technology

[0002] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most fundamental and important techniques in modern biological research, widely used for protein separation, purification, molecular weight determination, and subsequent Western blotting analysis. Its basic principle is based on the polymerization of polyacrylamide (PA) with the cross-linking agent N,N'-methylenebisacrylamide (Bis) to form a three-dimensional network structure with specific pore sizes. Under the influence of an electric field, negatively charged proteins bound to SDS migrate within the gel network according to their molecular weight, thus achieving separation.

[0003] However, traditional polyacrylamide gels, especially low-concentration gels used for separating large molecular weight proteins (e.g., gels with a total monomer concentration (T%) below 8%), have significant mechanical property defects. They are typically soft, brittle, and lack elasticity, exhibiting extremely poor tensile and tear resistance. In practical operations, such as peeling the gel from a glass plate, transferring the gel in staining and destaining solutions, or during the "sandwich" assembly and transfer operations in Western blotting experiments, the gel is highly susceptible to breakage, defects, or even complete fracture. Gel damage not only leads to the loss of valuable samples and experimental data but also severely affects the morphology of electrophoretic bands and the accuracy of subsequent quantitative analysis, forcing researchers to repeat time-consuming and laborious experiments.

[0004] To overcome the insufficient mechanical strength of polyacrylamide gels, researchers have made numerous attempts. One common approach is to increase the proportion of the cross-linking agent (Bis-acrylamide, Bis), but this often results in an overly stiff and brittle gel with increased opacity, potentially affecting protein migration and resolution. Another approach is to introduce a second polymer network, such as preparing polyacrylamide-agarose composite gels. While the addition of agarose can improve gel strength, agarose requires heating to dissolve and then solidifying at room temperature, complicating the preparation process. Furthermore, the macroporous structure of agarose can significantly alter the gel's sieving properties, hindering the separation of small molecular weight proteins. In recent years, studies have also attempted to introduce nanomaterials (such as nanoclays and carbon nanotubes) or synthesize polymers to enhance hydrogels, but these materials are often costly and may present biocompatibility issues or interfere with subsequent optical analysis.

[0005] Hydroxymethyl cellulose (HMC) is a nonionic ether derivative of cellulose. As a naturally sourced polysaccharide polymer, HMC possesses excellent water solubility, biocompatibility, film-forming properties, and thickening properties. In materials science, cellulose derivatives are frequently used to improve the rheological and mechanical properties of materials. However, detailed studies on the specific use of HMC to enhance the mechanical strength of SDS-PAGE gels while maintaining their electrophoretic separation performance have not yet been reported.

[0006] Therefore, developing a method that can significantly improve the mechanical strength and ease of handling of SDS-PAGE gels without compromising their protein separation resolution and optical transparency is of great practical significance for improving the efficiency and data quality of biological experiments. Summary of the Invention

[0007] The purpose of this invention is to solve the technical problems of low mechanical strength, fragility, and difficulty in handling of SDS-PAGE gels, especially low-concentration gels, in the prior art, and to provide a high mechanical strength SDS-PAGE gel containing hydroxymethyl cellulose, its preparation method, and its application.

[0008] The first aspect of the present invention provides a composition for preparing SDS-PAGE gel, the composition comprising: acrylamide, N,N'-methylenebisacrylamide, sodium dodecyl sulfate and hydroxymethyl cellulose, wherein the weight-average molecular weight of the hydroxymethyl cellulose is 50,000 Da to 1,000,000 Da; and the components are stored separately and not mixed with each other before use.

[0009] Preferably, the composition further includes a buffer reagent and an initiator system.

[0010] Preferably, the buffer reagent is Tris-HCl, and the initiator is the system APS / TEMED.

[0011] A second aspect of the present invention provides a high mechanical strength SDS-PAGE gel containing hydroxymethyl cellulose, wherein the SDS-PAGE gel is prepared by polymerization of at least acrylamide monomer, N,N'-methylenebisacrylamide, sodium dodecyl sulfate (SDS), a buffer reagent, an initiator system, and hydroxymethyl cellulose, wherein the weight-average molecular weight of the hydroxymethyl cellulose is from 50,000 Da to 1,000,000 Da, and the final concentration of the hydroxymethyl cellulose in the final gel system is from 0.05% (w / v) to 2.0% (w / v).

[0012] More preferably, the final concentration of the hydroxymethyl cellulose in the final gel system is 0.1% (w / v) to 0.8% (w / v).

[0013] Preferably, the hydroxymethyl cellulose is physically dispersed or entangled in a crosslinked polymer network formed by acrylamide and N,N'-methylenebisacrylamide.

[0014] Preferably, the total monomer concentration (T%) of the SDS-PAGE gel is 4% to 20%, and the degree of crosslinking (C%) is 1% to 5%.

[0015] Preferably, the mass ratio of acrylamide to bisacrylamide is 29:1.

[0016] A third aspect of the present invention provides a method for preparing the aforementioned SDS-PAGE gel, comprising: S1: Preparation of hydroxymethyl cellulose stock solution: Dissolve hydroxymethyl cellulose powder in deionized water and stir until completely dissolved to obtain a transparent or translucent hydroxymethyl cellulose stock solution; S2: Preparation of gel premix: Mix acrylamide / N,N'-methylenebisacrylamide stock solution, Tris-HCl buffer, SDS solution, deionized water and hydroxymethyl cellulose stock solution prepared in step S1; S3: Polymerization: Add the initiator system to the gel premix obtained in step S2, mix well, pour into a gel mold, and polymerize to form a gel.

[0017] Preferably, step S1 further includes: first wetting the hydroxymethyl cellulose powder with ethanol and then adding it to deionized water, or sprinkling the hydroxymethyl cellulose powder into vigorously stirred deionized water, thereby fully dissolving the hydroxymethyl cellulose.

[0018] A fourth aspect of the present invention provides a method for separating and identifying proteins based on the above-described SDS-PAGE gel, the method comprising using the SDS-PAGE gel as a separating gel or simultaneously as a stacking gel.

[0019] Preferably, the separation and identification of proteins includes protein gel electrophoresis analysis, membrane transfer operation in Western blotting assay, and in-gel enzyme activity analysis.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Significantly enhanced mechanical strength and toughness: The addition of hydroxymethyl cellulose significantly improves the tensile strength, elongation at break, and tear resistance of SDS-PAGE gels. The gel becomes more elastic and less prone to breakage during operations such as peeling, transfer, staining, and decolorization, greatly facilitating experimental procedures and increasing the success rate of experiments. The enhancing effect is particularly significant for fragile low-concentration gels (such as 8% or 10% separating gels).

[0021] (2) Maintaining excellent electrophoretic performance: Within the preferred concentration range, the addition of hydroxymethylcellulose will not interfere with the normal migration of proteins in SDS-PAGE, and the protein bands will remain sharp and clear, with a resolution comparable to the control gel without added hydroxymethylcellulose.

[0022] (3) Maintaining good optical transparency: Hydroxymethyl cellulose and polyacrylamide system have good compatibility, and the prepared composite gel maintains high optical transparency, which does not affect gel imaging and optical density scanning analysis.

[0023] (4) Low cost and simple preparation: Hydroxymethyl cellulose is an inexpensive and readily available industrial raw material. The preparation method of the present invention only requires adding a step of adding hydroxymethyl cellulose solution to the conventional gelling process, without the need for special equipment or complex chemical modifications, and is easy to promote and apply in ordinary laboratories.

[0024] (5) Good biocompatibility: Hydroxymethyl cellulose is a non-toxic and biocompatible material that will not have an adverse effect on subsequent biological experiments (such as protein spectrum analysis and activity detection). Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Mechanical strength analysis of SDS-PAGE separating gels with and without hydroxymethyl cellulose at different concentrations.

[0027] Figure 2 Image showing the staining results of protein bands obtained using an 8% SDS-PAGE separating gel without hydroxymethyl cellulose.

[0028] Figure 3 Image showing the staining results of protein bands obtained using an 8% SDS-PAGE separating gel containing 0.5% hydroxymethylcellulose.

[0029] Figure 4Image showing the staining results of protein bands obtained using a 10% SDS-PAGE separating gel containing 0.2% hydroxymethylcellulose.

[0030] Figure 5 Transmittance analysis of SDS-PAGE separating gels with and without hydroxymethyl cellulose at different concentrations. Detailed Implementation

[0031] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0032] The first aspect of this invention provides a high mechanical strength SDS-PAGE gel containing hydroxymethyl cellulose. The SDS-PAGE gel is mainly polymerized from acrylamide monomer, N,N'-methylenebisacrylamide, sodium dodecyl sulfate (SDS), buffer reagent, initiator system and reinforcing agent, wherein the reinforcing agent is physically dispersed or entangled in a cross-linked polymer network formed by acrylamide and cross-linking agent.

[0033] The buffer reagent is Tris-HCl, the initiator is the system APS / TEMED, and the enhancer is hydroxymethylcellulose.

[0034] This invention is based on the design concept of a semi-interpenetrating polymer network (semi-IPN). In this application, pre-dissolved linear hydroxymethyl cellulose (HMC) is introduced during the free radical polymerization of acrylamide and a crosslinking agent (Bis) to form a chemically crosslinked network. The long-chain HMC molecules are physically interwoven and entangled within the three-dimensional polyacrylamide network. More importantly, the abundant hydroxyl groups (-OH) on the HMC molecular chains can form numerous intermolecular hydrogen bonds with the amide groups (-CONH2) on the polyacrylamide chains.

[0035] This physical entanglement and extensive hydrogen bond network constitute a second layer of physical cross-linking. When the gel is subjected to external stretching or compression, these physical interactions (especially the breaking and reforming of hydrogen bonds) can effectively dissipate energy and prevent the propagation of microcracks, thereby significantly improving the gel's toughness, tensile strength, and tear resistance. Since hydroxymethyl cellulose (HMC) is non-ionic and has good hydrophilicity, the appropriate addition of HMC does not significantly alter the internal charge environment of the gel, nor does it cause significant phase separation leading to gel turbidity. Therefore, it can maintain the high resolution and transparency required for SDS-PAGE.

[0036] In this invention, the concentration of hydroxymethyl cellulose in the final gel system is a crucial parameter. Too low a concentration (e.g., below 0.05%) results in minimal enhancement; too high a concentration (e.g., above 2.0%) leads to excessive viscosity of the premix, making gel casting and degassing difficult. Furthermore, an overly dense hydroxymethyl cellulose network may hinder the migration of large protein molecules, resulting in band dispersion or decreased resolution.

[0037] Preferably, the final concentration of the hydroxymethyl cellulose in the final gel system is from 0.05% (w / v) to 2.0% (w / v).

[0038] More preferably, the final concentration of the hydroxymethyl cellulose in the final gel system is 0.1% (w / v) to 0.8% (w / v).

[0039] The inventors discovered that the molecular weight of the hydroxymethyl cellulose also affects the reinforcing effect and solution viscosity. Generally, the higher the molecular weight, the stronger the thickening and entanglement effect, but the more difficult it is to dissolve.

[0040] Preferably, the weight-average molecular weight of the hydroxymethyl cellulose is from 50,000 Da to 1,000,000 Da.

[0041] Preferably, the total monomer concentration (T%) of the SDS-PAGE gel is 4% to 20%, and the degree of crosslinking (C%) is 1% to 5%.

[0042] Preferably, the mass ratio of acrylamide to bisacrylamide is 29:1.

[0043] A second aspect of the present invention provides a method for preparing the aforementioned SDS-PAGE gel, comprising: preparing a hydroxymethyl cellulose stock solution, preparing a gel premix, and polymerizing it into a gel.

[0044] The hydroxymethyl cellulose stock solution is dissolved using either the "wetting method" (wetting the powder with a small amount of ethanol before adding water) or the "sprinkling method" (slowly sprinkling the powder into a vigorously stirred water vortex) to prevent the hydroxymethyl cellulose powder from clumping together when it comes into contact with water.

[0045] The inventors discovered that whether hydroxymethyl cellulose is added only to the separating gel of a conventional SDS-PAGE gel formulation system, or added to both the stacking gel and the separating gel, the overall strength of the gel can be improved.

[0046] The third aspect of this invention provides an application of the aforementioned SDS-PAGE gel in the fields of protein analysis.

[0047] Preferably, the applications include protein gel electrophoresis analysis, membrane transfer operations in Western blotting assays, and in-gel enzyme activity analysis.

[0048] The following description is based on specific embodiments: Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available, and techniques not described in detail were performed according to standard methods well known to those skilled in the art.

[0049] The main reagents and instruments used in this invention are shown in Table 1: Table 1 List of Reagents and Instruments The general-purpose stock solution used in this invention is prepared as follows: 30% Acr / Bis solution (29:1): Weigh 29g acrylamide and 1g bisacrylamide, dissolve them in ultrapure water and bring the volume to 100mL. After filtration, store in a brown bottle at 4℃.

[0050] 1.5 M Tris-HCl (pH 8.8): Weigh 18.1 g Tris and dissolve it in 60 mL of ultrapure water. Adjust the pH to 8.8 with HCl and add water to a final volume of 100 mL. Used as a separating gel buffer.

[0051] 1.0 M Tris-HCl (pH 6.8): Weigh 12.1 g Tris and dissolve it in 60 mL of ultrapure water. Adjust the pH to 6.8 with HCl and add water to a final volume of 100 mL. Used as a stacking gel buffer.

[0052] 10% SDS solution: Weigh 10g of SDS and dissolve it in 100mL of ultrapure water.

[0053] 10% APS solution: Weigh 10g of APS and dissolve it in 100mL of ultrapure water. Prepare fresh solution immediately before use.

[0054] Preparation of 2% (w / v) carboxymethyl cellulose (HMC) stock solution: Weigh 2.0 g of carboxymethyl cellulose powder. Add approximately 80 mL of ultrapure water to a beaker and turn on a magnetic stirrer until a deep vortex is generated. Slowly sprinkle the carboxymethyl cellulose powder into the center of the vortex, avoiding clumping. Continue stirring until the powder is completely dissolved, forming a homogeneous, transparent or slightly opalescent viscous solution. Dilute to 100 mL with water. Store this solution at 4°C; allow it to reach room temperature and mix well before use.

[0055] Example 1: Preparation of 10% SDS-PAGE separating gel containing 0.2% hydroxymethyl cellulose Prepare according to the formula described in Table 2.

[0056] Table 2 Separating Gel Formulation (10 mL) The specific preparation steps of the separating gel are as follows: S1: In a clean small beaker or centrifuge tube, add ultrapure water, 2% HMC stock solution, 30% Acr / Bis solution, 1.5 M Tris-HCl buffer and 10% SDS solution in sequence, and mix gently to avoid generating too many bubbles.

[0057] S2: Add 10% APS solution and TEMED, and mix quickly and gently.

[0058] S3: Immediately pour the mixture into the pre-assembled vertical electrophoresis glass plate interlayer to the appropriate height.

[0059] S4: Lightly cover the rubber surface with a layer of water or isopropyl alcohol to isolate oxygen and flatten the rubber surface.

[0060] S5: Let stand at room temperature for about 30-60 minutes until the gel is fully polymerized.

[0061] S6: Remove the capping layer, rinse the glue surface with ultrapure water, and then pour the concentrate according to the conventional method (the concentrate may or may not contain hydroxymethyl cellulose; in this embodiment, the concentrate does not contain hydroxymethyl cellulose).

[0062] Comparative Example 1: Preparation of 10% SDS-PAGE separating gel without hydroxymethyl cellulose The difference from Example 1 is that an equal volume of ddH2O was used instead of the 2% HMC stock solution in Example 1.

[0063] Example 2: Preparation of 8% SDS-PAGE separating gel containing 0.5% hydroxymethyl cellulose Prepare according to the formula described in Table 3.

[0064] Table 3. Separating gel formulation (10 mL) The preparation steps are the same as in Example 1. During dispensing, the solution may feel more viscous than ordinary colloids; therefore, slow operation is necessary to prevent air bubbles from accumulating.

[0065] Example 3: Preparation of SDS-PAGE separating gel containing hydroxymethyl cellulose In this embodiment, a 2% HMC stock solution with a weight-average molecular weight of 1,000,000 Da was used, and the other parameters were the same as in Example 2.

[0066] Comparative Example 2: Preparation of 8% SDS-PAGE separating gel without hydroxymethyl cellulose The difference from Example 2 is that an equal volume of ddH2O was used instead of the 2% HMC stock solution in Example 2.

[0067] Performance testing and effect evaluation To verify the effectiveness of the present invention, the gels prepared in the examples and comparative examples were subjected to the following tests: 1. Evaluation of mechanical operation feel (qualitative test) Blind testing was conducted by experienced laboratory personnel. After gel polymerization, routine procedures such as peeling off the glass plate, handling the gel, and transferring it to a tray were performed.

[0068] Comparative Example 1 and Comparative Example 2 (without hydroxymethyl cellulose): The 10% gel felt hard but brittle, and small cracks easily appeared at the edges when peeled off; the control gel with a concentration of 8% or lower was very soft and easily broke when handled.

[0069] Example 1 (0.2% hydroxymethyl cellulose) and Examples 2 and 3 (0.5% hydroxymethyl cellulose): The gel exhibited significant toughness and elasticity. The feel was no longer "brittle and hard," but rather had a texture similar to cartilage. It was not easily broken when gently pulled in the hand, nor was it easily damaged when handled with tweezers. The enhancing effect of Example 2 was more significant than that of Example 1.

[0070] 2. Mechanical strength test The mechanical strength of protein gels without added hydroxymethyl cellulose (HMC) and with different concentrations of HMC was tested. The prepared gels were cut into standard dumbbell-shaped strips and subjected to uniaxial tensile tests (tensile rate 10 mm / min) on a universal testing machine. The maximum force (N) required at the moment of fracture was recorded to directly characterize the macroscopic mechanical strength of the gel. The gel matrix used in the experiment was a 30% acrylamide-bisacrylamide solution (Acr-Bis, 29:1). 0%, 0.2%, and 0.5% HMC were added to this matrix to investigate the enhancing effect of HMC on the mechanical properties of the gel.

[0071] Test results are as follows Figure 1 As shown, the results indicate that the tensile strength of the gel increases significantly with the increase of the amount of hydroxymethyl cellulose (HMC), indicating that the introduction of HMC can significantly improve the load-bearing capacity of the gel, and the reinforcing effect is positively correlated with the concentration of HMC. These results confirm that introducing a small amount of HMC (0.2%-0.5%) into the acrylamide-bisacrylamide gel system can increase the tensile strength of the gel by more than 30%, demonstrating that HMC can effectively enhance the mechanical stability and tensile properties of the gel network.

[0072] 3. Electrophoretic separation performance test The same protein molecular weight standards (markers) and whole protein samples were loaded onto the gels prepared in Examples 1, 2, and Comparative Example 2. Electrophoresis was performed under standard conditions (constant voltage 300V) until the bromophenol blue indicator reached the bottom. After staining and destaining with iBlue™ PAGE rapid staining solution, the samples were photographed and analyzed using an LED light-emitting plate. The protein band staining results obtained from the 8% SDS-PAGE separating gel without hydroxymethyl cellulose are shown below. Figure 2 As shown, the staining results of protein bands obtained from an 8% SDS-PAGE separating gel containing 0.5% hydroxymethyl cellulose are as follows. Figure 3 As shown, the staining results of protein bands obtained from a 10% SDS-PAGE separating gel containing 0.2% hydroxymethyl cellulose are as follows. Figure 4 As shown.

[0073] The stained gel images show that in the biogels of Examples 1 and 2, the protein bands are clear, sharp, and flat, without diffusion, tailing, or distortion, and the band resolution is good. This indicates that adding hydroxymethyl cellulose to the SDS-PAGE gel within the preferred concentration range of this invention does not change the effective sieving pore size distribution of the gel, nor does it hinder the normal electrophoretic migration of proteins. It perfectly preserves the inherent high-resolution separation function of the SDS-PAGE gel and meets the experimental requirements of routine protein electrophoretic separation.

[0074] 4. Optical transparency test The fully polymerized gel sheets prepared in Examples 1, 2, and 2 were placed in a spectrophotometer, and the transmittance of each gel sheet at a visible light wavelength of 400 nm was measured using ultrapure water as a blank reference. At the same time, the transparency of the gel was observed by the naked eye to evaluate whether it met the experimental requirements for conventional photography and optical density scanning analysis.

[0075] The test results are as follows Figure 5 As shown, the transmittance of Comparative Example 2 is above 98%. The transmittance of Examples 1 and 2 both remain above 97%, and the gels are still highly transparent to the naked eye, fully meeting the requirements for routine photography and optical density scanning analysis.

[0076] In summary, this invention successfully constructed a semi-interpenetrating network gel by simply introducing an appropriate amount of hydroxymethyl cellulose into a conventional SDS-PAGE gel formulation. Experimental results strongly demonstrate that this modified gel maintains excellent protein electrophoretic separation performance and optical transparency while fundamentally improving its mechanical strength and toughness. This enhancement significantly reduces the risk of gel breakage during experimental operations, improves the reliability and success rate of experiments, and possesses good practical value and application prospects.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for preparing SDS-PAGE gel, the composition comprising: acrylamide, N,N'-methylenebisacrylamide, sodium dodecyl sulfate and hydroxymethyl cellulose, wherein the weight-average molecular weight of the hydroxymethyl cellulose is from 50,000 Da to 1,000,000 Da; and the components are stored separately and not mixed with each other before use.

2. The composition according to claim 1, characterized in that, It also includes buffer reagents and initiator systems.

3. The composition according to claim 2, characterized in that, The buffer reagent is Tris-HCl, and the initiator is the system APS / TEMED.

4. An SDS-PAGE gel, characterized in that, It is prepared by polymerization of at least acrylamide, N,N'-methylenebisacrylamide, sodium dodecyl sulfate, buffer reagent, initiator system and hydroxymethyl cellulose, wherein the hydroxymethyl cellulose has a weight-average molecular weight of 50,000 Da to 1,000,000 Da and a final concentration of 0.05% (w / v) to 2.0% (w / v) in the final gel system.

5. The SDS-PAGE gel according to claim 4, characterized in that, The hydroxymethyl cellulose is physically dispersed or entangled in a cross-linked polymer network formed by acrylamide and N,N'-methylenebisacrylamide.

6. The SDS-PAGE gel according to claim 4, characterized in that, The total monomer concentration of the SDS-PAGE gel is 4% to 20%, and the degree of crosslinking is 1% to 5%.

7. The SDS-PAGE gel according to claim 4, characterized in that, The mass ratio of the acrylamide to the bisacrylamide is 29:

1.

8. A method for preparing an SDS-PAGE gel according to any one of claims 4-7, comprising: S1: Preparation of hydroxymethyl cellulose stock solution: Dissolve hydroxymethyl cellulose powder in deionized water and stir until completely dissolved to obtain a transparent or translucent hydroxymethyl cellulose stock solution; S2: Preparation of gel premix: Mix acrylamide / N,N'-methylenebisacrylamide stock solution, Tris-HCl buffer, SDS solution, deionized water and hydroxymethyl cellulose stock solution prepared in step S1; S3: Polymerization: Add the initiator system to the gel premix obtained in step S2, mix well, pour into a gel mold, and polymerize to form a gel.

9. The method according to claim 8, characterized in that, Step S1 further includes: first wetting the hydroxymethyl cellulose powder with ethanol and then adding it to deionized water, or sprinkling the hydroxymethyl cellulose powder into vigorously stirred deionized water, thereby fully dissolving the hydroxymethyl cellulose.

10. A method for separating and identifying proteins using an SDS-PAGE gel according to any one of claims 4-7, the method comprising using the SDS-PAGE gel as a separating gel or simultaneously as a stacking gel.

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