Rapid film transfer method

Abstract

The invention belongs to the technical field of biotechnology and protein determination, and particularly relates to a rapid membrane transfer method. The method comprises the following steps: firstly, soaking gel in a pretreatment solution, sequentially soaking a solid-phase membrane in methanol and an equilibrium solution, then assembling a membrane transfer device, adding a membrane transfer buffer solution, and transferring the membrane by adopting a gradient electric field. Through the synergistic effect of gel pretreatment, optimization of a membrane transfer buffer solution formula and a gradient electric field membrane transfer mode, the membrane transfer time is shortened, and the experiment efficiency is greatly improved. According to the membrane transfer method, the transfer efficiency of proteins with the molecular weight of 10-200 kDa reaches 90% or above, the problems that high-molecular-weight proteins are incompletely transferred and small-molecular-weight proteins are prone to penetrating in an existing method are solved, the sensitivity and accuracy of subsequent detection are remarkably improved, and the membrane transfer method is suitable for large-scale application and popularization.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and protein assay technology, and specifically relates to a rapid membrane transfer method. Background Technology

[0002] Western blotting is a core technology in biotechnology for detecting specific proteins. Its key steps include SDS-PAGE gel electrophoresis separation of protein samples, transfer of proteins from the gel to a solid membrane (transfer), blocking of non-specific sites on the membrane, specific binding of primary antibody to the target protein, binding of secondary antibody to primary antibody, and signal detection. Among these, the transfer step serves as a bridge between gel separation and membrane detection; its efficiency and quality directly affect the accuracy, sensitivity, and experimental cycle of subsequent detection results.

[0003] Currently, commonly used transfer methods mainly include wet transfer, semi-dry transfer, and dry transfer. Wet transfer, with its advantages of high transfer efficiency, clear protein bands, and wide applicability (suitable for transferring proteins of various molecular weights), has become the most commonly used transfer method in laboratories. However, its core drawback lies in its excessively long transfer time. For proteins with typical molecular weights (20-100 kDa), the transfer time usually requires 1-2 hours, while for high molecular weight proteins (>100 kDa), the transfer time can even exceed 4 hours, severely limiting experimental efficiency, especially in scenarios requiring high-throughput sample detection or urgent experiments.

[0004] Semi-dry transfer shortens the transfer time (about 30-60 minutes for conventional protein transfer) by reducing the buffer distance between the gel and the membrane and decreasing the resistance to current conduction. However, this method has problems such as unstable transfer efficiency, incomplete transfer of high molecular weight proteins, and the tendency for bands to tail or become blurred. In addition, it has strict requirements on the formulation and concentration of the buffer and has a low tolerance for operational errors.

[0005] Although dry transfer is the fastest method (about 10-20 minutes), the equipment is expensive, and proteins are prone to degradation or denaturation during the transfer process. In addition, it is only suitable for the transfer of small molecular weight proteins (<50kDa), which has a narrow scope of application and cannot meet the detection needs of complex samples.

[0006] To address the core contradiction in existing transfer methods—the difficulty of balancing efficiency and quality—researchers have made numerous attempts. For example, they have improved transfer speed by optimizing the ionic strength of the transfer buffer, adding methanol substitutes (such as ethanol or isopropanol), and adjusting parameters like transfer voltage or current. However, these improvements often only achieve a slight reduction in transfer time (e.g., 20%-30% reduction in wet transfer), and are prone to problems such as incomplete protein transfer and band diffusion. Other studies have attempted to improve transfer efficiency by using novel solid-phase membrane materials or improving the structure of transfer devices, but these efforts are limited by material performance or equipment cost, making large-scale application difficult.

[0007] Further analysis of existing transfer techniques reveals that the essence of the transfer process is the movement and adsorption of proteins from the gel interior to the solid membrane surface under the influence of an electric field, overcoming the resistance of the gel matrix and the ion migration resistance of the buffer solution. In existing methods, the transfer buffer has a single component (the conventional formulation is Tris, glycine, and methanol), which has limited ion conductivity, protein solubility, and gel pore expansion capacity, resulting in the electric field not being able to effectively act on the proteins inside the gel. Simultaneously, the use of a constant voltage or current during transfer makes it impossible to dynamically adjust the electric field strength according to the protein molecular weight distribution and gel thickness. This leads to small protein molecules being easily over-transferred (penetrating the solid membrane), while large protein molecules struggle to overcome gel resistance for complete transfer. Furthermore, the Joule heating generated during transfer causes the buffer temperature to rise, which not only accelerates protein denaturation but also increases the buffer's resistance, further reducing transfer efficiency.

[0008] Therefore, developing a rapid transfer method that combines fast transfer speed, high transfer efficiency, good strip quality, wide applicability, and simple operation is of great practical significance and application value for promoting the development of protein detection technology. Summary of the Invention

[0009] To address the problems existing in the prior art, this invention proposes a rapid transfer method. Through the synergistic effect of gel pretreatment, optimized transfer buffer formulation, and gradient electric field transfer mode, the transfer time is shortened, significantly improving experimental efficiency. This invention's transfer method achieves a transfer efficiency of over 90% for proteins with molecular weights of 10-200 kDa, solving the problems of incomplete transfer of high molecular weight proteins and easy penetration of small molecular weight proteins in existing methods. This significantly improves the sensitivity and accuracy of subsequent detection, making it suitable for large-scale application.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a transfer film method, the transfer film method comprising the following steps: (1) Gel pretreatment: Immerse the gel after SDS-PAGE electrophoresis in pretreatment solution, shake at room temperature, and rinse with deionized water 2-3 times after removal; (2) Solid phase membrane pretreatment: Immerse the PVDF membrane or nitrocellulose membrane in methanol (this step can be omitted for nitrocellulose membrane), and then transfer it to the equilibrium solution for immersion; (3) Assembly of the transfer device: Assemble the transfer sandwich in the order of “cathode plate → sponge pad → filter paper → gel → solid membrane → filter paper → sponge pad → anode plate”, ensuring that there are no air bubbles between each layer. After assembly, place it in the transfer tank. During the assembly of the transfer sandwich, the size of the filter paper is consistent with the size of the gel and solid membrane, and each layer is aligned to avoid misalignment that could lead to uneven transfer. (4) Adding transfer buffer: Add pre-cooled transfer buffer to the transfer tank at 4°C. Avoid generating air bubbles during the addition process and ensure that the buffer completely submerges the transfer sandwich. (5) Gradient electric field transfer: Place the transfer tank in a 4℃ environment and connect the power supply to perform gradient electric field transfer; the transfer process is divided into three stages: the first stage voltage is set to 25-30V and lasts for 5-8 minutes; the second stage voltage is set to 35-40V and lasts for 8-12 minutes; the third stage voltage is set to 20-25V and lasts for 3-5 minutes; the total transfer time is 16-25 minutes. (6) Post-transfer processing: After the transfer is completed, remove the solid membrane and rinse it 2-3 times with TBST buffer. Then block, incubate with primary antibody, incubate with secondary antibody and detect signal.

[0011] Further, in step (1), the pretreatment solution is composed of the following components in parts by weight: 10-15 parts Tris, 0.5-1.5 parts EDTA, 8-12 parts urea, 0.3-0.8 parts Tween-20, and 80-90 parts deionized water.

[0012] Furthermore, the pretreatment solution is composed of the following components in parts by weight: 12 parts Tris, 1.0 part EDTA, 10 parts urea, 0.5 parts Tween-20, and 86.5 parts deionized water.

[0013] The role of urea is to disrupt the hydrogen bond structure of the gel, expand the gel pore size, and reduce the migration resistance of proteins within the gel; Tween-20 can improve the solubility of proteins and reduce the adsorption of proteins on the gel surface.

[0014] Furthermore, in step (2), the methanol soaking time is 1-2 minutes, and the equilibrium solution soaking time is 5-8 minutes.

[0015] Furthermore, both the equilibration solution and the transfer buffer are composed of the following components in parts by weight: 8-12 parts Tris, 30-40 parts glycine, 5-10 parts ethylene glycol, 2-5 parts polyethylene glycol-6000, 0.8-1.5 parts sodium dodecyl sulfate, 15-25 parts methanol, and 70-80 parts deionized water.

[0016] Furthermore, both the equilibration solution and the transfer buffer are composed of the following components in parts by weight: 10 parts Tris, 35 parts glycine, 8 parts ethylene glycol, 3 parts polyethylene glycol-6000, 1.2 parts sodium dodecyl sulfate, 20 parts methanol, and 75 parts deionized water.

[0017] Furthermore, the mass ratio of ethylene glycol to polyethylene glycol-6000 is 2-3:1. The synergistic effect of the two can lower the freezing point of the buffer solution, improve ion conduction efficiency, and reduce protein denaturation. The sodium dodecyl sulfate can enhance the charge of proteins and improve the driving effect of electric field on proteins.

[0018] Furthermore, the voltage for the first stage of the transfer process is set to 28V for 6 minutes; the voltage for the second stage is set to 38V for 10 minutes; the voltage for the third stage is set to 22V for 4 minutes; and the total transfer time is 20 minutes.

[0019] Furthermore, during the transfer process, the temperature of the transfer buffer solution in the transfer tank is controlled at 4-8°C, and the temperature is maintained by a circulating cooling device or an ice bath.

[0020] Furthermore, the PVDF membrane has a pore size of 0.45 μm and is suitable for proteins with a molecular weight >20 kDa, or a pore size of 0.22 μm and is suitable for proteins with a molecular weight <20 kDa, and the nitrocellulose membrane preferably has a pore size of 0.45 μm.

[0021] Furthermore, the transfer method is applicable to the transfer of proteins with a molecular weight of 10-200kDa. For proteins with a molecular weight >150kDa, the voltage of the second stage in step (5) is increased to 42V, the duration is extended to 15 minutes, and the total transfer time is adjusted to 25-30 minutes.

[0022] The present invention has the following beneficial effects: (1) Significantly improved transfer speed: This invention reduces the total transfer time to 16-25 minutes through the synergistic effect of gel pretreatment, optimized transfer buffer formulation and gradient electric field transfer mode. Compared with the traditional wet transfer method (1-2 hours), the transfer efficiency is increased by more than 70%, and compared with the semi-dry transfer method (30-60 minutes), the transfer time is reduced by more than 40%, which greatly improves the experimental efficiency.

[0023] (2) High transfer efficiency and wide applicability: Urea in the gel pretreatment solution of this invention can expand the gel pore size, EDTA can chelate metal ions to prevent protein degradation, and Tween-20 can improve protein solubility; ethylene glycol, PEG 6000 and SDS added to the transfer buffer work synergistically to enhance ion conduction efficiency and protein charge; the gradient electric field mode can dynamically adjust the electric field strength according to the protein migration characteristics to ensure that small molecular weight proteins do not penetrate the membrane and large molecular weight proteins are completely transferred. Experiments have shown that the method of this invention achieves a transfer efficiency of over 90% for proteins with molecular weights of 10-200 kDa, solving the problems of incomplete transfer of high molecular weight proteins and easy penetration of small molecular weight proteins in existing methods.

[0024] (3) Excellent band quality: The proportion of methanol in the transfer buffer of this invention is optimized to 15-25 parts, which can reduce the non-specific binding of protein to gel while maintaining the denaturation state of protein; the combination of gradient electric field and low temperature environment (4-8℃) can effectively reduce Joule heat generation, avoid protein band diffusion, tailing or deformation, and the protein band after transfer is clear, with neat edges and no obvious background interference, which significantly improves the sensitivity and accuracy of subsequent detection.

[0025] (4) Simple operation and controllable cost: The reagents used in this invention are all conventional laboratory reagents, requiring no special equipment or expensive materials. The membrane transfer device is the same as the traditional wet transfer method, requiring no additional modification. The operation process is simple and easy to learn, making it suitable for large-scale promotion and application.

[0026] (5) High stability: By strictly controlling the mass fraction of each component and the transfer parameters, this invention ensures the repeatability of the transfer effect. Multiple experiments have shown that when different batches and different operators use the method of this invention for transfer, the coefficient of variation of the transfer efficiency is less than 5%, and the strip quality is consistent, which solves the problem of unstable transfer effect in existing methods. Detailed Implementation

[0027] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0029] Example 1 A rapid transfer method, the specific steps of which are as follows: (1) Gel pretreatment: The gel after SDS-PAGE electrophoresis (containing standard protein marker, BSA, β-actin, histone H3, myosin) was immersed in the pretreatment solution and shaken at 120 rpm for 8 minutes at room temperature. After removal, it was rinsed 3 times with deionized water. The pretreatment solution was composed of the following components in parts by weight: Tris 12 parts, EDTA 1.0 parts, urea 10 parts, Tween-20 0.5 parts, and deionized water 86.5 parts. (2) Solid phase membrane pretreatment: The 0.45 μm PVDF membrane was immersed in methanol for 1.5 minutes, and then transferred to the equilibration buffer for 6 minutes; the equilibration buffer had the same composition as the transfer buffer. (3) Assembly of the transfer device: Assemble the transfer sandwich in the order of “cathode plate → sponge pad → filter paper → gel → PVDF membrane → filter paper → sponge pad → anode plate”, ensuring no air bubbles, and place it in the transfer tank; (4) Adding transfer buffer: Add pre-cooled transfer buffer to the transfer tank to completely submerge the transfer sandwich; the transfer buffer is composed of the following components in parts by weight: Tris 10 parts, glycine 35 parts, ethylene glycol 8 parts, polyethylene glycol-6000 3 parts, SDS 1.2 parts, methanol 20 parts, and deionized water 75 parts; (5) Gradient electric field transfer: Place the transfer tank in a low temperature environment of 4℃ and connect the power supply to perform gradient electric field transfer; the first stage voltage is 28V and lasts for 6 minutes; the second stage voltage is 38V and lasts for 10 minutes; the third stage voltage is 22V and lasts for 4 minutes; the total transfer time is 20 minutes. (6) Post-transfer processing: Take out the PVDF membrane, rinse it 3 times with TBST buffer, and then block, incubate with primary antibody, incubate with secondary antibody and perform ECL chemiluminescence detection.

[0030] Example 2 A rapid transfer method, the specific steps of which are as follows: (1) Gel pretreatment: The gel after SDS-PAGE electrophoresis was immersed in the pretreatment solution and shaken at 100 rpm for 10 minutes at room temperature. After removal, it was rinsed twice with deionized water. The pretreatment solution consisted of the following components in parts by weight: 15 parts Tris, 1.5 parts EDTA, 12 parts urea, 0.8 parts Tween-20, and 80 parts deionized water. (2) Solid membrane pretreatment: The 0.22 μm PVDF membrane was immersed in methanol for 2 minutes, and then transferred to the equilibration buffer for 8 minutes; the equilibration buffer had the same composition as the transfer buffer. (3) Assembly of the film transfer device: Same as in Example 1; (4) Adding transfer buffer: Add pre-cooled transfer buffer to the transfer tank to completely submerge the transfer sandwich; the transfer buffer is composed of the following components in parts by weight: Tris 12 parts, glycine 40 parts, ethylene glycol 10 parts, polyethylene glycol-6000 5 parts, SDS 1.5 parts, methanol 25 parts, and deionized water 70 parts; (5) Gradient electric field transfer: Place the transfer tank in a low temperature environment of 4℃ and connect the power supply to perform gradient electric field transfer; the first stage voltage is 30V and lasts for 8 minutes; the second stage voltage is 40V and lasts for 12 minutes; the third stage voltage is 25V and lasts for 5 minutes; the total transfer time is 25 minutes. (6) Post-transfer treatment: Same as in Example 1.

[0031] Example 3 A rapid transfer method, the specific steps of which are as follows: (1) Gel pretreatment: The gel after SDS-PAGE electrophoresis was immersed in the pretreatment solution and shaken at 150 rpm for 5 minutes at room temperature. After removal, it was rinsed 3 times with deionized water. The pretreatment solution consisted of the following components in parts by weight: 10 parts Tris, 0.5 parts EDTA, 8 parts urea, 0.3 parts Tween-20, and 90 parts deionized water. (2) Solid membrane pretreatment: The 0.45 μm nitrocellulose membrane was directly transferred to the equilibration solution and immersed for 5 minutes; the equilibration solution had the same composition as the transfer buffer. (3) Assembly of the film transfer device: Same as in Example 1; (4) Adding transfer buffer: Add pre-cooled transfer buffer to the transfer tank to completely submerge the transfer sandwich; the transfer buffer is composed of the following components in parts by weight: 8 parts Tris, 30 parts glycine, 5 parts ethylene glycol, 2 parts polyethylene glycol-6000, 0.8 parts SDS, 15 parts methanol, and 80 parts deionized water; (5) Gradient electric field transfer: Place the transfer tank in a low temperature environment of 4℃ and connect the power supply to perform gradient electric field transfer; the first stage voltage is 25V and lasts for 5 minutes; the second stage voltage is 35V and lasts for 8 minutes; the third stage voltage is 20V and lasts for 3 minutes; the total transfer time is 16 minutes. (6) Post-transfer treatment: Same as in Example 1.

[0032] Comparative Example 1 (Missing Gel Pretreatment Steps) A transfer method, except that the gel pretreatment in step 1 is omitted, is completely identical to that in Example 1.

[0033] Comparative Example 2 (Urea was missing in the pretreatment solution) A membrane transfer method, in which the composition of the pretreatment solution is adjusted to: 12 parts Tris, 1.0 part EDTA, 0.5 parts Tween-20, and 96.5 parts deionized water (excluding urea), and the remaining steps are completely consistent with those in Example 1.

[0034] Comparative Example 3 (pretreatment solution lacking Tween-20) A transfer membrane method, in which the composition of the pretreatment solution is adjusted to: 12 parts Tris, 1.0 part EDTA, 10 parts urea, and 97 parts deionized water (excluding Tween-20), and the remaining steps are completely consistent with those in Example 1.

[0035] Comparative Example 4 (Ethylene glycol was missing from the transfer buffer) A transfer method, in which the components of the transfer buffer are adjusted as follows: 10 parts Tris, 35 parts glycine, 3 parts polyethylene glycol-6000, 1.2 parts SDS, 20 parts methanol, and 83 parts deionized water (excluding ethylene glycol), and the remaining steps are completely consistent with those in Example 1.

[0036] Comparative Example 5 (PEG 6000 missing in transfer buffer) A transfer method, in which the components of the transfer buffer are adjusted as follows: 10 parts Tris, 35 parts glycine, 8 parts ethylene glycol, 1.2 parts SDS, 20 parts methanol, and 85 parts deionized water (PEG 6000 is missing), and the remaining steps are completely consistent with those in Example 1.

[0037] Comparative Example 6 (SDS missing in transfer buffer) A transfer method, in which the components of the transfer buffer are adjusted as follows: 10 parts Tris, 35 parts glycine, 8 parts ethylene glycol, 3 parts polyethylene glycol-6000, 20 parts methanol, and 86.2 parts deionized water (SDS is missing), and the remaining steps are completely consistent with those in Example 1.

[0038] Comparative Example 7 (using constant voltage film transfer, without gradient electric field) A transfer method, in which the transfer mode of step (5) is adjusted to: constant voltage 30V for 20 minutes (consistent with the total transfer time of Example 1), and the remaining steps are completely consistent with Example 1.

[0039] Comparative Example 8 (Traditional Wet Conversion Method) A traditional wet-conversion method, the specific steps are as follows: (1) Gel pretreatment: The gel after SDS-PAGE electrophoresis was immersed in conventional transfer buffer for equilibration for 10 minutes; the conventional transfer buffer was composed of the following components in parts by weight: Tris 3 parts, glycine 14 parts, methanol 20 parts, and deionized water 87 parts; (2) PVDF membrane pretreatment: Same as in Example 1; (3) Assembly of the film transfer device: Same as in Example 1; (4) Adding transfer buffer: Add conventional transfer buffer pre-cooled to 4°C to the transfer tank; (5) Transfer: constant voltage 100V for 90 minutes (conventional time for traditional wet transfer method). (6) Post-transfer treatment: Same as in Example 1.

[0040] Comparative Example 9 (Traditional Semi-Dry Conversion Method) A traditional semi-dry conversion method, the specific steps are as follows: (1) Pretreatment of gel and membrane: Same as steps (1) and (2) of Comparative Example 8; (2) Assembly of the transfer device: Assemble the semi-dry transfer device in the order of "cathode plate → filter paper → gel → PVDF membrane → filter paper → anode plate"; (3) Transfer: constant current 250mA, for 40 minutes (conventional time for traditional semi-dry transfer method). (4) Post-transfer treatment: Same as in Example 1.

[0041] Comparative Example 10 (the methanol content in the transfer buffer was 10 parts by mass, which is below the scope of this invention). A transfer method, in which the components of the transfer buffer are adjusted as follows: 10 parts Tris, 35 parts glycine, 8 parts ethylene glycol, 3 parts polyethylene glycol-6000, 1.2 parts SDS, 10 parts methanol, and 80 parts deionized water (10 parts by weight of methanol), and the remaining steps are completely consistent with those in Example 1.

[0042] Comparative Example 11 (the methanol content in the transfer buffer was 30 parts by mass, which is higher than the scope of this invention). A transfer method, in which the components of the transfer buffer are adjusted as follows: 10 parts Tris, 35 parts glycine, 8 parts ethylene glycol, 3 parts polyethylene glycol-6000, 1.2 parts SDS, 30 parts methanol, and 75 parts deionized water (30 parts by weight of methanol). The remaining steps are completely consistent with those in Example 1.

[0043] Comparative Example 12 (The voltage in the first stage of the gradient electric field is 20V, which is below the scope of this invention). A transfer method is described, in which the gradient electric field transfer parameters are adjusted as follows: first stage voltage 20V, lasting 6 minutes; second stage voltage 38V, lasting 10 minutes; third stage voltage 22V, lasting 4 minutes; total transfer time 20 minutes, and the remaining steps are completely consistent with those in Example 1.

[0044] Test case 1. Transfer efficiency detection (Coomassie brilliant blue staining method + gray value analysis) Transfer efficiency reflects the proportion of proteins that migrate from the gel to the solid membrane and is a core indicator for evaluating the transfer effect. The detection steps are as follows: (1) Sample preparation: All examples and comparative examples used the same batch of SDS-PAGE gels, and the same mass (20 μg / lane) of mixed protein samples (including standard protein marker, BSA, β-actin, histone H3, myosin) were loaded onto the gels to ensure that the protein content of each lane was consistent.

[0045] (2) Transfer operation: The transfer process was completed according to the methods of each example and comparative example. After the transfer was completed, the gel (residual untransferred protein) and the solid membrane (successfully transferred protein) were collected respectively.

[0046] (3) Gel staining and destaining: Place the gel after transfer into Coomassie Brilliant Blue R-250 staining solution and stain by shaking at room temperature for 30 minutes (shaking speed 100 rpm); after staining, discard the staining solution, add Coomassie Brilliant Blue destaining solution, and destain by shaking at room temperature. Change the destaining solution 2-3 times during the process, 15 minutes each time, until the gel background is transparent and the protein bands are clearly visible.

[0047] (4) Membrane staining and destaining: Rinse the solid membrane after transfer with deionized water 3 times for 2 minutes each time to remove the residual transfer buffer on the surface; then put the membrane into Coomassie Brilliant Blue R-250 staining solution and stain with shaking at room temperature for 15 minutes (shaking speed 80 rpm); after staining, discard the staining solution, add Coomassie Brilliant Blue destaining solution, and destain with shaking at room temperature. Change the destaining solution 1-2 times for 10 minutes each time until the membrane background is transparent and the protein bands are clear.

[0048] (5) Image acquisition: The decolorized gel and solid membrane were placed in the gel imaging system and the same light intensity (5000 lux), exposure time (1s) and resolution (300dpi) were used to capture the images to ensure that the images were free of reflection and distortion.

[0049] (6) Gray value analysis: The collected images of the gel and membrane were opened using ImageJ software. First, background correction was performed (using the "Rolling Ball" algorithm with a radius of 50 pixels). Then, the band regions corresponding to each molecular weight protein were selected (residual bands on the gel and transfer bands on the membrane). The integrated density value (IDV) of each band was recorded.

[0050] (7) Calculation of transfer efficiency: Transfer efficiency (%) = (IDV of a certain molecular weight protein band on the membrane / (IDV of the residual protein band of the same molecular weight on the gel + IDV of the protein band of the same molecular weight on the membrane)) × 100%. Each example and comparative example were tested three times, and the mean ± standard deviation was taken as the final result.

[0051] 2. Strip clarity detection (chemiluminescence method + signal-to-noise ratio analysis) Band clarity reflects the sharpness of protein bands and the level of background interference. It is quantitatively evaluated using the signal-to-noise ratio (SNR). The detection steps are as follows: (1) Post-transfer treatment: After the transfer is completed, remove the solid membrane and rinse it three times with TBST buffer for 5 minutes each time to remove the residual transfer buffer.

[0052] (2) Blocking: Place the membrane in a 5% skim milk powder blocking solution and shake at room temperature for 1 hour (shaking speed 100 rpm) to block non-specific binding sites.

[0053] (3) Primary antibody incubation: Discard the blocking solution, rinse the membrane 3 times with TBST buffer for 5 minutes each time; then add the diluted primary antibody (anti-BSA antibody, anti-β-actin antibody, anti-histone H3 antibody, and anti-myosin antibody diluted in the blocking solution at a volume ratio of 1:1000), and incubate overnight at 4°C with shaking (shaking speed 50 rpm).

[0054] (4) Secondary antibody incubation: Discard the primary antibody incubation solution, rinse the membrane 3 times with TBST buffer for 10 minutes each time, add diluted HRP-labeled secondary antibody (diluted in blocking solution at a volume ratio of 1:5000), and incubate at room temperature with shaking for 1 hour (shaking speed 100 rpm).

[0055] (5) Signal detection: Discard the secondary antibody incubation solution, rinse the membrane 3 times with TBST buffer for 10 minutes each time; mix solution A and solution B of the ECL chemiluminescence kit at a volume ratio of 1:1, and add them evenly to the membrane surface, incubate at room temperature for 2 minutes; then place the membrane into the chemiluminescence imaging system, use the automatic exposure mode (exposure time range 10s-5min) to acquire the strip signal image, and ensure that the signal intensity is within the detection linear range (avoid overexposure or underexposure).

[0056] (6) Signal-to-noise ratio (SNR) calculation: The signal image was opened using ImageJ software. First, the band regions of proteins of each molecular weight were selected, and the average signal intensity of the bands was recorded. Then, the same area of ​​background region with no signal near the bands was selected, and the average signal intensity of the background was recorded. SNR = average signal intensity of the bands / average signal intensity of the background. Each example and comparative example was tested three times, and the mean ± standard deviation was taken as the final result.

[0057] 3. Evaluation of band integrity (morphological observation method) Band integrity reflects the regularity of protein band morphology, with no tailing, diffusion, or breakage. The evaluation steps are as follows: (1) Image acquisition: Use the chemiluminescence signal image acquired in the above "strip clarity detection" to ensure that the image is clear and without blur.

[0058] (2) Morphological observation: Using the magnification function of the chemiluminescence imaging system (magnification 10×), observe the edge morphology, width uniformity, and whether there are phenomena such as tailing, diffusion, and breakage of protein bands of each molecular weight.

[0059] (3) Grading: Based on the observation results, the grades are divided into four levels: Advantages: The strip edges are neat and sharp, the width is uniform, and there is no trailing, diffusion or breakage. There is no cross-contamination between the strips. Good: The strip edges are basically neat, the width is uniform, there is a very slight trailing (the trailing length is less than 1 / 5 of the strip width), and there is no diffusion or breakage. Medium: The strip edges are blurred, the width is uneven, there is obvious tailing (the tail length is 1 / 5-1 / 3 of the strip width), slight diffusion, and the strip is basically intact without breakage; Poor: The edges of the strips are severely blurred, the width varies significantly, there is severe tailing (tail length > 1 / 3 of the strip width), obvious diffusion, and some strips are broken.

[0060] Each embodiment and comparative example was independently evaluated by three professional experimentalists, and the consistent results were taken as the final grade; if there were any differences, a consensus was reached through consultation.

[0061] 4. Repeatability test (calculation of coefficient of variation) Repeatability reflects the stability of the method under the same conditions, and is evaluated by calculating the coefficient of variation (CV) of the transfer efficiency. The detection steps are as follows: (1) Parallel experiments: Three parallel samples were set up for each example and comparative example, and the transfer and transfer efficiency were tested under the same experimental conditions (same batch of reagents, same instrument, same operator).

[0062] (2) Data statistics: Record the transfer efficiency test results of 3 parallel samples (with 42kDa β-actin as the representative index), and calculate the mean and standard deviation (SD).

[0063] (3) Calculation of coefficient of variation: CV = (SD / Mean) × 100%. The smaller the CV value, the better the repeatability of the method.

[0064] The test data of the membrane transfer method in Examples 1-3 obtained according to the above methods are shown in Table 1 below.

[0065] Table 1 project Example 1 Example 2 Example 3 Transfer time (minutes) 20 25 16 Transfer efficiency (%) -10kDa 92.3±1.2 93.5±0.9 90.8±1.5 Transfer efficiency (%) -42kDa 95.6±0.8 96.2±0.7 94.3±1.1 Transfer efficiency (%) -66kDa 94.8±1.0 95.5±0.6 93.7±1.3 Transfer efficiency (%) -150kDa 91.5±1.4 92.8±1.1 89.6±1.6 Transfer efficiency (%) -200kDa 89.7±1.7 91.2±1.3 88.5±1.8 Strip SNR-10kDa 8.6±0.5 8.8±0.4 8.2±0.6 Strip SNR-42kDa 10.3±0.3 10.5±0.2 9.8±0.4 Strip SNR-66kDa 9.9±0.4 10.2±0.3 9.5±0.5 Strip SNR-150kDa 8.3±0.6 8.5±0.5 7.9±0.7 Strip SNR-200kDa 7.8±0.7 8.1±0.6 7.5±0.8 Strip integrity level excellent excellent good Transfer efficiency CV (%) 1.3±0.2 1.1±0.1 1.5±0.3 The test data of the transfer membrane method obtained in Comparative Examples 1-12 are shown in Table 2 below.

[0066] Table 2 Comparative numbering Transfer time (minutes) Transfer efficiency (%) Strip SNR Strip integrity level Transfer efficiency CV (%) Key Defect Analysis 1 20 78.5±2.3 6.2±0.8 middle 2.9±0.4 Without gel pretreatment, the gel pore size was not expanded, resulting in high transfer resistance. 2 20 82.3±1.9 6.8±0.7 middle 2.3±0.3 The pretreatment solution lacked urea, resulting in insufficient gel pore expansion. 3 20 80.1±2.1 6.5±0.9 middle 2.6±0.4 The pretreatment solution lacks Tween-20 and has poor protein solubility. 4 20 85.6±1.7 7.3±0.6 good 2.0±0.3 The transfer buffer is ethylene glycol-free, resulting in low ion conduction efficiency. 5 20 84.2±1.8 7.1±0.7 good 2.1±0.3 The transfer buffer lacks PEG 6000, resulting in poor protein stability. 6 20 83.7±1.9 7.0±0.8 good 2.3±0.4 The transfer buffer lacks SDS, resulting in insufficient protein charge. 7 20 81.5±2.2 6.7±0.9 middle 2.7±0.4 At constant voltage, small molecules can easily penetrate while large molecules transfer incompletely. 8 90 95.2±0.9 10.1±0.4 excellent 1.0±0.1 Excessive transfer time results in low efficiency. 9 40 86.3±2.0 7.5±0.6 good 2.3±0.3 Incomplete transfer of high molecular weight proteins resulted in slight band tailing. 10 20 87.8±1.6 7.6±0.5 good 1.8±0.2 Insufficient methanol leads to incomplete protein denaturation and poor membrane adsorption. 11 20 82.6±2.0 6.9±0.8 middle 2.4±0.3 Excessive methanol leads to excessive protein denaturation and band diffusion. 12 20 79.3±2.2 6.4±0.9 middle 2.8±0.4 The voltage was too low in the first stage, resulting in insufficient initial migration momentum. The comparative data of protein transfer efficiency of different molecular weights obtained from the tests of Example 1, Comparative Example 8 and Comparative Example 9 are shown in Table 3 below.

[0067] Table 3 Protein molecular weight (kDa) Example 1 (20 minutes) Comparative Example 8 (90 minutes, conventional wet rotation) Comparative Example 9 (40 minutes, traditional semi-dry rotation) 10 92.3±1.2 94.8±0.7 88.5±1.4 17 (Histone H3) 93.1±1.1 95.1±0.6 89.2±1.3 42 (β-actin) 95.6±0.8 95.2±0.9 86.3±2.0 66 (BSA) 94.8±1.0 94.9±0.8 85.7±1.8 150 91.5±1.4 93.6±1.0 78.9±2.5 200 (myosin) 89.7±1.7 92.3±1.2 75.6±2.8 The test results above show that: 1. As shown in Table 1, the transfer time of Examples 1-3 of the present invention is only 16-25 minutes, which is shorter than that of the traditional wet transfer method (90 minutes) and semi-dry transfer method (40 minutes). The transfer efficiency for proteins of different molecular weights (10-200kDa) all reach more than 88.5%, of which the transfer efficiency of conventional molecular weight proteins of 42kDa and 66kDa exceeds 93%, which is close to the level of the traditional wet transfer method. The band SNR is above 7.5, the band integrity level is good to excellent, and the coefficient of variation of transfer efficiency is less than 1.5%, indicating that the method of the present invention has the advantages of fast transfer speed, high transfer efficiency, good band quality, and strong reproducibility.

[0068] 2. Comparative Example 1, lacking the gel pretreatment step, had a transfer efficiency of only 78.5% and a band integrity of medium, indicating that gel pretreatment can effectively expand the gel pore size and improve protein solubility, which is a key step in improving transfer efficiency. The transfer efficiencies of Comparative Example 2 (without urea) and Comparative Example 3 (without Tween-20) were 82.3% and 80.1%, respectively, both lower than that of Example 1 (95.6%), proving that the gel pore expansion effect of urea and the protein solubilization effect of Tween-20 have a synergistic effect and are indispensable.

[0069] 3. The transfer efficiencies of Comparative Example 4 (deficient in ethylene glycol), Comparative Example 5 (deficient in PEG 6000), and Comparative Example 6 (deficient in SDS) were 85.6%, 84.2%, and 83.7%, respectively, all lower than those of Example 1. The band SNR also decreased significantly. This indicates that the synergistic effect of ethylene glycol, PEG 6000, and SDS in the transfer buffer can improve ion conduction efficiency, enhance protein charge, and maintain protein stability, which are the core factors for achieving rapid and efficient transfer.

[0070] 4. Comparative Example 7 used constant voltage for membrane transfer, with a transfer efficiency of 81.5% and medium band integrity. This indicates that the gradient electric field mode can dynamically adjust the electric field strength according to the protein migration characteristics, avoiding the problems of small molecule proteins penetrating the membrane and large molecule proteins being transferred incompletely. It has significant advantages over the constant voltage mode.

[0071] 5. As shown in Table 3, the transfer time of Example 1 is only 1 / 4.5 of that of the traditional wet transfer method and 1 / 2 of that of the traditional semi-dry transfer method, while the transfer efficiency is comparable to that of the traditional wet transfer method (the difference in transfer efficiency for proteins of conventional molecular weight is less than 1%), and significantly higher than that of the traditional semi-dry transfer method (especially for proteins with a molecular weight >150kDa, the difference in transfer efficiency exceeds 10%). The band SNR is close to that of the traditional wet transfer method and superior to that of the traditional semi-dry transfer method, proving that the method of the present invention can significantly shorten the transfer time while ensuring the transfer quality, and solve the contradiction between efficiency and quality in the traditional method.

[0072] 6. The transfer efficiencies of Comparative Example 10 (10 parts methanol) and Comparative Example 11 (30 parts methanol) were 87.8% and 82.6%, respectively, both lower than those of Example 1 (20 parts methanol). This indicates that the mass fraction of methanol in the transfer buffer needs to be controlled within the range of 15-25 parts. If it is too low, the protein denaturation will be insufficient and the membrane adsorption capacity will decrease. If it is too high, the protein will be excessively denatured, leading to band diffusion and affecting the transfer quality.

[0073] 7. In Comparative Example 12, the first-stage voltage was 20V (lower than the range of 25-30V in this invention), and the transfer efficiency was 79.3%, which was lower than that in Example 1. This indicates that the low voltage in the first stage will lead to insufficient initial migration momentum of the protein, which will not be able to quickly detach from the gel matrix, thus affecting the overall transfer efficiency. This proves the rationality of the gradient electric field parameters of this invention.

[0074] In summary, this invention achieves rapid and efficient protein transfer through the synergistic effect of gel pretreatment, optimized transfer buffer formulation, and gradient electric field transfer mode. The transfer time is only 1 / 2 to 1 / 4.5 of that of traditional methods, and the transfer efficiency and band quality are comparable to those of traditional wet transfer methods. It has a wide range of applications, strong reproducibility, and significant inventiveness and practical value.

[0075] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A transfer film method, characterized in that, The transfer method includes the following steps: (1) Gel pretreatment: The gel after SDS-PAGE electrophoresis was immersed in pretreatment solution and shaken at room temperature. After removal, it was rinsed with deionized water. (2) Solid phase membrane pretreatment: Immerse the PVDF membrane or nitrocellulose membrane in methanol, and then transfer it to the equilibrium solution for immersion; (3) Assembly of the transfer device: Assemble the transfer sandwich in the order of "cathode plate → sponge pad → filter paper → gel → solid membrane → filter paper → sponge pad → anode plate" to ensure that there are no air bubbles between each layer. After assembly, place it in the transfer tank. (4) Adding transfer buffer: Add pre-cooled transfer buffer to the transfer tank to ensure that the transfer sandwich is completely submerged in the buffer; (5) Gradient electric field transfer: Place the transfer tank in a 4℃ environment and connect the power supply to perform gradient electric field transfer; the transfer process is divided into three stages: the first stage voltage is set to 25-30V and lasts for 5-8 minutes; the second stage voltage is set to 35-40V and lasts for 8-12 minutes; the third stage voltage is set to 20-25V and lasts for 3-5 minutes; the total transfer time is 16-25 minutes. (6) Post-transfer processing: After the transfer is completed, the solid membrane is removed, rinsed with TBST buffer, and then blocked, incubated with primary antibody, incubated with secondary antibody and signal detection are performed.

2. The transfer film method according to claim 1, characterized in that, In step (1), the pretreatment solution is composed of the following components in parts by weight: 10-15 parts Tris, 0.5-1.5 parts EDTA, 8-12 parts urea, 0.3-0.8 parts Tween-20, and 80-90 parts deionized water.

3. The transfer film method according to claim 2, characterized in that, The pretreatment solution consists of the following components in parts by weight: 12 parts Tris, 1.0 part EDTA, 10 parts urea, 0.5 parts Tween-20, and 86.5 parts deionized water.

4. The transfer film method according to claim 1, characterized in that, In step (2), the methanol soaking time is 1-2 minutes, and the equilibrium solution soaking time is 5-8 minutes.

5. The transfer film method according to claim 1, characterized in that, The equilibration solution and transfer buffer are each composed of the following components in parts by weight: Tris 8-12 parts, glycine 30-40 parts, ethylene glycol 5-10 parts, polyethylene glycol-6000 2-5 parts, sodium dodecyl sulfate 0.8-1.5 parts, methanol 15-25 parts, and deionized water 70-80 parts.

6. The transfer film method according to claim 5, characterized in that, The equilibration solution and transfer buffer are each composed of the following components in parts by weight: Tris 10 parts, glycine 35 parts, ethylene glycol 8 parts, polyethylene glycol-6000 3 parts, sodium dodecyl sulfate 1.2 parts, methanol 20 parts, and deionized water 75 parts.

7. The transfer film method according to claim 1, characterized in that, The voltage for the first stage of the transfer process is set to 28V for 6 minutes; the voltage for the second stage is set to 38V for 10 minutes; the voltage for the third stage is set to 22V for 4 minutes; and the total transfer time is 20 minutes.

8. The transfer film method according to claim 1, characterized in that, During the transfer process, the temperature of the transfer buffer solution in the transfer tank is controlled at 4-8℃, and the temperature is maintained by a circulating cooling device or an ice bath.

9. The transfer film method according to claim 1, characterized in that, The PVDF membrane has a pore size of 0.45 μm and is suitable for proteins with a molecular weight >20 kDa, or a pore size of 0.22 μm and is suitable for proteins with a molecular weight <20 kDa. The nitrocellulose membrane preferably has a pore size of 0.45 μm.

10. The transfer method according to claim 1, characterized in that, The transfer method is suitable for the transfer of proteins with a molecular weight of 10-200kDa. For proteins with a molecular weight >150kDa, the voltage of the second stage in step (5) is increased to 42V and the duration is extended to 15 minutes, and the total transfer time is adjusted to 25-30 minutes.