Use of a polyvinylidene fluoride microporous filter membrane and a western blotting method

Abstract

The application belongs to the technical field of protein transfer printing, and provides a use of a polyvinylidene fluoride microporous filter membrane and a Western blot method, the polyvinylidene fluoride microporous filter membrane has spherulites and rod-like short fiber crystals on the surface and does not have a dense skin layer, the use includes that the polyvinylidene fluoride microporous filter membrane is used as a solid carrier to detect a microgram-level protein sample with high sensitivity in a Western blot process, and clear protein bands are obtained, the polyvinylidene fluoride microporous filter membrane is prepared by the following method: at a first temperature, polyvinylidene fluoride is dissolved in a solvent and an additive is added, a mixture is obtained to obtain a feed liquid, the feed liquid is cooled to a second temperature to obtain a casting solution; the casting solution is prepared to form a liquid membrane; after steam-induced phase separation of the liquid membrane, the liquid membrane is immersed in a coagulation bath to be coagulated and formed, and the polyvinylidene fluoride microporous filter membrane is prepared. The polyvinylidene fluoride microporous filter membrane prepared by the application has good protein transfer printing effect in the Western blot process, and the improvement of the detection effect is particularly significant.

Description

Technical Field

[0001] This application relates to the field of protein transfer technology, and in particular to the use of a polyvinylidene fluoride microporous filter membrane and a protein immunoblotting method. Background Technology

[0002] Western blotting is a technique that transfers proteins onto a membrane, which are then detected using antibodies. It is commonly used to identify specific proteins and to perform qualitative and semi-quantitative analysis.

[0003] Commonly used solid-phase supports in protein immunoblotting include polyvinylidene fluoride (PVDF) membranes and nitrocellulose (NC) membranes. NC membranes are relatively inexpensive and widely used, but their binding strength and toughness are weaker than PVDF membranes, and they cannot be reused. However, they have a high protein adsorption capacity and good hydrophilicity. PVDF membranes, on the other hand, offer advantages such as higher mechanical strength, lower background, wider solvent compatibility, and better staining ability, and can also be used for protein sequencing.

[0004] However, PVDF membranes are hydrophobic and require pre-wetting with anhydrous methanol to impart hydrophilicity. Furthermore, existing techniques for fabricating PVDF membranes result in unclear protein transfer bands and insufficient sensitivity.

[0005] Currently, PVDF membranes with excellent protein transfer performance are mainly manufactured abroad. These membranes undergo hydrophilic modification of PVDF, resulting in complex and costly preparation processes. Domestically produced PVDF membranes rarely achieve the same protein transfer performance as foreign membranes when used for Western blotting. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides an application of polyvinylidene fluoride microporous filter membrane and a protein immunoblotting method to solve the problems in the prior art where the protein transfer effect of domestically prepared PVDF membranes in protein immunoblotting is not as good as that of foreign-prepared PVDF membranes, and the cost is high.

[0007] To achieve the above and related objectives, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of polyvinylidene fluoride microporous filter membrane as a solid-phase carrier for protein immunoblotting, comprising: using polyvinylidene fluoride microporous filter membrane as a solid-phase carrier in the process of protein immunoblotting to perform high-sensitivity detection of protein samples at the microgram level and obtain clear protein bands.

[0009] Polyvinylidene fluoride microporous filter membranes are prepared by the following method:

[0010] (1) At the first temperature, polyvinylidene fluoride is dissolved in a solvent and additives are added and mixed to obtain a liquid. The liquid is then cooled to the second temperature to obtain a casting solution.

[0011] (2) Prepare a liquid film from the casting solution;

[0012] (3) After the liquid membrane is subjected to steam-induced phase separation, it is immersed in a coagulation bath to solidify and form a polyvinylidene fluoride microporous filter membrane.

[0013] In one embodiment of this application, the first temperature is 40–80°C; and / or, the second temperature is 30–60°C.

[0014] In one embodiment of this application, the mass fraction of polyvinylidene fluoride in the liquid in step (1) is 10-30 wt%.

[0015] And / or, the solvent mass fraction is 50–80 wt%;

[0016] And / or, the mass fraction of the additive is 0–20 wt%.

[0017] In one embodiment of this application, the solvent includes one or more of N,N-dimethylacetamide, N,N-methylformamide, N-methyl-2-pyrrolidone, triethyl phosphate, and 2-pyrrolidone.

[0018] In one embodiment of this application, the additive includes one or more of methanol, ethanol, glycerol, isopropanol, n-butanol, diethylene glycol, triethylene glycol, tetraethylene glycol, ethylene glycol methyl ether, ethylene glycol dimethyl ether, and water.

[0019] In one embodiment of this application, step (3) includes: placing the liquid membrane in a low-temperature and low-humidity environment for vapor-induced phase separation, wherein the temperature of the low-temperature and low-humidity environment is 10-30°C and the relative humidity is 40-80%RH.

[0020] In one embodiment of this application, the steam induction time in step (3) is 120 to 600 s.

[0021] In one embodiment of this application, the second temperature is further preferably 30-50°C.

[0022] In one embodiment of this application, step (3) includes: immersing in a coagulation bath to solidify and form a membrane, washing, drying, and obtaining a polyvinylidene fluoride microporous filter membrane.

[0023] A second aspect of the present invention provides a protein immunoblotting method, comprising: separating and transferring a protein sample by electrophoresis on a solid support, and finally detecting and analyzing it by antigen-antibody reaction, wherein the solid support is a polyvinylidene fluoride microporous filter membrane having the above-mentioned uses.

[0024] The beneficial technical effects of this invention are as follows:

[0025] This invention first lowers the temperature of the feed solution to ensure that the feed solution system is in a metastable state; then, under low temperature and low humidity conditions, non-solvent vapor is used to induce crystallization and phase separation in the membrane, generating spherulites and rod-shaped short fiber crystals; finally, the membrane is immersed in a coagulation bath to exchange out the solvent and other components, thereby obtaining a polyvinylidene fluoride microporous filter membrane.

[0026] The polyvinylidene fluoride microporous filter membrane prepared by this invention using a simple process can be used as a solid-phase carrier in a short-time and rapid protein immunoblotting process to perform highly sensitive detection of protein samples at the microgram level, and obtain clear protein bands. The protein transfer effect is good, and the improvement in detection effect is particularly significant.

[0027] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0028] The accompanying drawings, incorporated in and forming part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application. It is obvious that the drawings described below are merely some embodiments of this application, and those skilled in the art can obtain other drawings based on these drawings without inventive effort. In the drawings:

[0029] Figure 1 Here is an electron microscope image of the surface of the PVDF microporous filter membrane in Example 1;

[0030] Figure 2 Here is an electron microscope image of the surface of the PVDF microporous filter membrane in Example 2;

[0031] Figure 3 Here is an electron microscope image of the surface of the PVDF microporous filter membrane in Example 3;

[0032] Figure 4 Electron micrograph of the surface of the PVDF microporous filter membrane in Comparative Example 1;

[0033] Figure 5 Electron micrograph of the surface of the 2PVDF microporous filter membrane (comparative example);

[0034] Figure 6 This is an electron microscope image of the surface of a 3PVDF microporous filter membrane, which is a comparative example.

[0035] Figure 7 This is a protein band marker distribution diagram of the PVDF microporous filter membrane used in Example 1 for GADPH transfer testing;

[0036] Figure 8This is a protein band marker distribution diagram of the protein transfer test of Escherichia coli supernatant using PVDF microporous filter membrane in Example 1;

[0037] Figure 9 This is a marker distribution diagram of protein bands for testing the sensitivity of different existing transfer membranes to the target protein.

[0038] Figure 10 This is a protein band marker distribution diagram for the sensitivity test of the PVDF microporous filter membrane and the existing transfer membrane for the target protein in Example 1. Detailed Implementation

[0039] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be understood that certain features of the invention (described in the context of separate embodiments for clarity) may also be provided in combination in a single embodiment. Conversely, multiple features of the invention (described in the context of a single embodiment for brevity) may also be provided separately or in any suitable combination or, where appropriate, in any other described embodiment of the invention. Certain features described in the context of various embodiments will not be considered essential features of those embodiments unless the embodiment is inoperable without those elements. The invention is further illustrated below by specific examples; however, it should be noted that the specific process conditions and results described in the embodiments of the invention are merely illustrative and should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be covered within the scope of protection of the invention.

[0040] First, it should be noted that the raw materials used in the technical solution of this invention, including polyvinylidene fluoride (PVDF), N,N-dimethylacetamide (DMAC), N,N-methylformamide (DMF), N-methyl-2-pyrrolidone (NMP), triethyl phosphate (TEP), 2-pyrrolidone, methanol, ethanol, glycerol, isopropanol, n-butanol, diethylene glycol, triethylene glycol, tetraethylene glycol, ethylene glycol methyl ether, and ethylene glycol dimethyl ether, are all commercially available products.

[0041] This invention provides the use of polyvinylidene fluoride microporous filter membrane as a solid-phase carrier for protein immunoblotting, comprising: using polyvinylidene fluoride microporous filter membrane as a solid-phase carrier in the process of protein immunoblotting to perform high-sensitivity detection of protein samples at the microgram level and obtain clear protein bands.

[0042] Polyvinylidene fluoride microporous filter membranes are prepared by the following method:

[0043] (1) At the first temperature, polyvinylidene fluoride is dissolved in a solvent and additives are added. The mixture is stirred and mixed evenly to obtain a liquid. The liquid is then cooled to the second temperature and kept warm to obtain a casting solution.

[0044] In this step, the first temperature is 40-80℃, preferably 50-70℃;

[0045] In this step, the second temperature is 30-60°C, preferably 30-50°C;

[0046] In this step, the mass fraction of polyvinylidene fluoride in the feed solution is 10-30 wt%, preferably 15-25 wt%.

[0047] In this step, the mass fraction of the solvent is 50-80 wt%, preferably 50-60 wt%.

[0048] In this step, the mass fraction of the additive is 0-20 wt%, preferably 10-20 wt%.

[0049] In this step, the solvent includes one or more of N,N-dimethylacetamide, N,N-methylformamide, N-methyl-2-pyrrolidone, triethyl phosphate, and 2-pyrrolidone;

[0050] In this step, the additives include one or more of methanol, ethanol, glycerol, isopropanol, n-butanol, diethylene glycol, triethylene glycol, tetraethylene glycol, ethylene glycol methyl ether, ethylene glycol dimethyl ether, and water.

[0051] (2) The casting solution is coated onto the PES release film, and the film is scraped to form a liquid film. The scraping distance is 200-500μm, preferably 300-400μm.

[0052] (3) The liquid membrane is placed in a low-temperature and low-humidity environment for vapor-induced phase separation. The temperature of the low-temperature and low-humidity environment is 10-30℃ and the relative humidity is 40-80RH.

[0053] In this step, the preferred temperature is 20–30°C;

[0054] In this step, the relative humidity is preferably 60-80% RH;

[0055] In this step, the steam induction time is 120–600 s, preferably 180–300 s.

[0056] (4) The liquid membrane after steam-induced phase separation is immersed in a coagulation bath to exchange solvent and additives, coagulate and form, clean, and dry to obtain polyvinylidene fluoride microporous filter membrane.

[0057] In this step, the coagulation bath temperature is 20–60°C, preferably 20–40°C;

[0058] In this step, the coagulation bath treatment time is 2 to 10 minutes, preferably 4 to 8 minutes;

[0059] In this step, the cleaning time is 5 to 20 minutes, preferably 10 to 20 minutes;

[0060] In this step, the drying temperature is 50–90°C, preferably 60–80°C;

[0061] In this step, the drying time is 1 to 10 minutes, preferably 2 to 8 minutes.

[0062] The present invention also provides a protein immunoblotting method, comprising: separating and transferring a protein sample by electrophoresis on a solid support, and finally detecting and analyzing it by antigen-antibody reaction, wherein the solid support is a polyvinylidene fluoride microporous filter membrane having the above-mentioned uses.

[0063] The present invention will be described in detail below through specific examples and embodiments. It should also be understood that the following embodiments are only for specific illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0064] Example 1

[0065] (1) Dissolve 60g of polyvinylidene fluoride in 180g of N,N-dimethylacetamide and add 60g of ethylene glycol dimethyl ether. Stir at 70°C for 8 hours until the polyvinylidene fluoride is evenly dissolved to obtain the liquid.

[0066] The liquid material is cooled to 30°C and kept at that temperature to obtain the casting solution.

[0067] (2) The casting solution is coated onto the PES release film, and the film is scraped to form a liquid film with a scraping distance of 300 μm.

[0068] (3) The liquid membrane was placed in a low-temperature and low-humidity environment for vapor-induced phase separation for 180s. The temperature of the low-temperature and low-humidity environment was 25℃ and the relative humidity was 80%RH.

[0069] (4) The liquid membrane after steam-induced phase separation is immersed in a coagulation bath at 25°C. The coagulation bath is pure water. After treatment for 8 minutes, it is solidified and formed to obtain a gel membrane.

[0070] The gel membrane was washed with pure water for 10 minutes and then dried at 70°C for 8 minutes to obtain a polyvinylidene fluoride microporous filter membrane.

[0071] Example 2

[0072] (1) Dissolve 54g of polyvinylidene fluoride in 216g of N,N-dimethylacetamide, add 27g of ethylene glycol dimethyl ether and 3g of water, stir at 80℃ for 8h until the polyvinylidene fluoride is evenly dissolved to obtain the liquid.

[0073] The liquid material is cooled to 40°C and kept at that temperature to obtain the casting solution.

[0074] (2) The casting solution is coated onto the PES release film, and the film is scraped to form a liquid film with a scraping distance of 350 μm.

[0075] (3) The liquid membrane was placed in a low-temperature and low-humidity environment for vapor-induced phase separation for 240s. The temperature of the low-temperature and low-humidity environment was 20℃ and the relative humidity was 60RH.

[0076] (4) The liquid membrane after steam-induced phase separation is immersed in a coagulation bath at 30°C. The coagulation bath is pure water. After treatment for 5 minutes, it is solidified and formed to obtain a gel membrane.

[0077] The gel membrane was washed with pure water for 5 minutes and then dried at 80°C for 5 minutes to obtain a polyvinylidene fluoride microporous filter membrane.

[0078] Example 3

[0079] (1) Dissolve 60g of polyvinylidene fluoride in 180g of N,N-dimethylacetamide, add 30g of ethylene glycol dimethyl ether and 30g of diethylene glycol, stir at 70°C for 8h until the polyvinylidene fluoride is evenly dissolved to obtain the liquid.

[0080] The liquid material is cooled to 30°C and kept at that temperature to obtain the casting solution.

[0081] (2) The casting solution is coated onto the PES release film, and the film is scraped to form a liquid film with a scraping distance of 350 μm.

[0082] (3) The liquid membrane was placed in a low-temperature and low-humidity environment for vapor-induced phase separation for 240s. The temperature of the low-temperature and low-humidity environment was 20℃ and the relative humidity was 70RH.

[0083] (4) The liquid membrane after steam-induced phase separation is immersed in a coagulation bath at 40°C. The coagulation bath is pure water. After 10 minutes of treatment, it is solidified and formed to obtain a gel membrane.

[0084] The gel membrane was washed with pure water for 10 minutes and then dried at 80°C for 10 minutes to obtain a polyvinylidene fluoride microporous filter membrane.

[0085] Comparative Example 1

[0086] The difference between this comparative example and Example 1 is as follows:

[0087] (1) Dissolve 60g of polyvinylidene fluoride in 180g of N,N-dimethylacetamide and add 60g of ethylene glycol dimethyl ether. Stir at 70°C for 8 hours until the polyvinylidene fluoride is evenly dissolved to obtain the casting solution.

[0088] Comparative Example 2

[0089] The difference between this comparative example and Example 1 is as follows:

[0090] (3) The liquid membrane was placed in a high temperature and high humidity environment (relative conditions) for vapor-induced phase separation for 180s. The temperature of the high temperature and high humidity environment was 35℃ and the relative humidity was 85RH.

[0091] Comparative Example 3

[0092] The difference between this comparative example and Example 1 is as follows:

[0093] (1) Dissolve 60g of polyvinylidene fluoride in 180g of N,N-dimethylacetamide and stir at 70℃ for 8h until the polyvinylidene fluoride is evenly dissolved to obtain the liquid.

[0094] The liquid material is cooled to 30°C and kept at that temperature to obtain the casting solution.

[0095] Performance testing

[0096] Bubble point pressure and flux: The bubble point pressure (test liquid: anhydrous ethanol) and flux (test liquid: anhydrous ethanol) of the membranes prepared in Examples 1-3 and Comparative Examples 1-3 were tested according to GB / T32361-2015 "Test Method for Pore Size of Separation Membranes: Bubble Point and Average Flow Rate Method". The test results are shown in Table 1.

[0097] Protein adsorption capacity: The protein adsorption capacity of the membranes prepared in Examples 1-3 and Comparative Examples 1-3 was determined using the following methods:

[0098] The membrane cross-section was determined by the sample fracturing in liquid nitrogen, and the surface to be observed was gold-coated in a vacuum environment using a sputtering coating instrument. After processing, the sample was scanned and imaged under an accelerating voltage of 10 kV. The dried transfer membrane was then placed in a protein solution of a certain concentration for static adsorption for 24 hours. The change in absorbance of the solution before and after adsorption was measured, and the amount of protein adsorbed by the membrane was calculated. The results are shown in Table 1.

[0099] Microstructure: The microstructure of the membranes prepared in Examples 1-3 and Comparative Examples 1-3 was observed using a scanning electron microscope, such as... Figures 1-6 As shown.

[0100] Protein transfer experiment:

[0101] I. The following experiments were conducted using the PVDF microporous filter membrane prepared in Example 1:

[0102] 1. Membrane wetting:

[0103] S1. Wet the dry film in alcohol (>50% methanol, ethanol or isopropanol) for 10-20 seconds, or until it changes from opaque white to uniform translucent gray;

[0104] S2. Immerse the membrane in ultrapure water for 1-2 minutes to displace the alcohol;

[0105] S3. Equilibrate the membrane in transfer buffer for 2–3 minutes or until it is ready for use.

[0106] 2. Transfer:

[0107] S1. Disperse the protein mixture on a polyacrylamide gel. The target protein is GAPDH. The protein mixture is the supernatant protein solution of HEK-293 cell lysis.

[0108] S2. Immerse the gel in transfer buffer and allow it to equilibrate for 10–15 minutes;

[0109] S3. Assemble the transfer stack according to the manufacturer's instructions for the transfer device used;

[0110] S4. Transfer the protein according to the instructions of the transfer equipment manufacturer;

[0111] S5. Remove the blot from the transfer system and briefly rinse the membrane in ultrapure water to remove gel debris. The blot can be air-dried and stored or used immediately for immunoassay steps.

[0112] 3. Immunological testing (standard immunological testing):

[0113] S1. If the imprint has dried, rewet it in alcohol (>50% methanol, ethanol or isopropanol) for 15 seconds or until it changes from opaque white to translucent gray;

[0114] S2. Rinse the imprint in ultrapure water for 1 minute;

[0115] S3. Place the blot in blocking buffer and incubate for 1 hour with gentle stirring. Dilute the primary antibody in commercially available antibody dilution buffer, wash buffer, or blocking buffer;

[0116] S4. Place the blot in a diluted primary antibody solution and incubate at room temperature for 1 hour (or overnight at 4°C) with gentle stirring;

[0117] S5. Use washing buffer (add) Dilute the secondary antibody with surfactant buffer (TBST or PBST) 3-5 times, 5 minutes each time. Prepare commercially available antibody dilution buffer and wash or blocking buffer to dilute the secondary antibody.

[0118] S6. Place the blot in a diluted enzyme-labeled secondary antibody solution and incubate at room temperature for 1 hour;

[0119] S7. Wash the blot 3-5 times with washing buffer, 5 minutes each time;

[0120] S8. Place the blot membrane in a clean container and add appropriate detection reagents;

[0121] S9. Incubate for 1–5 minutes according to the manufacturer’s instructions for the test reagent;

[0122] S10. For HRP or AP chemiluminescent reagents, expose the blot to X-ray film or acquire an image using a digital imaging system. For colorimetric detection, add the reagent and wait for the signal to appear.

[0123] The protein band marker distribution diagram of the PVDF microporous filter membrane in Example 1 for GADPH transfer test, obtained based on the above experimental methods, is shown below. Figure 7 As shown.

[0124] II. Using *E. coli* lysate as the target protein, with protein concentration gradients of 20 μg / L, 4 μg / L, and 0.8 μg / L, protein transfer experiments were performed according to the above experimental procedures. The protein band marker distribution map of the protein transfer test of *E. coli* supernatant using the PVDF microporous membrane in Example 1 was obtained, as shown below. Figure 8 As shown.

[0125] III. Nine cell lines were selected: HeLa, HEK-293, Jurkat, K-562, HepG2, L02, HSC-T6, ROS1728, and NIH / 3T3. SRP9 was used as the target protein. The transfer membranes of control group 1 and control group 2 (the transfer membrane product described in the applicant's prior patent application CN202311130714.X) were used for protein transfer experiments according to the above experimental steps. The experimental results are as follows: Figure 9 As shown.

[0126] IV. Nine cell lines were selected: U2OS, A549, HeLa, HEK-293, HepG2, K-562, HSC-T6, NIH / 3T3, and 4T1. FNTB was used as the target protein. Protein transfer experiments were performed using the PVDF microporous membrane from Example 1 and the transfer membrane from Control Group 1 (an existing hydrophilic modified transfer membrane) according to the experimental procedures described above. The experimental results are as follows: Figure 10 As shown.

[0127] The experimental data and analysis are as follows:

[0128] Table 1 shows the properties of the membranes prepared in Examples 1-3 and Comparative Examples 1-3.

[0129]

[0130] As shown in Table 1, the bubble point pressures of the PVDF microporous filter membranes in Examples 1 to 3 of this application are all lower than those of the PVDF membranes in Comparative Examples 1 to 3. This indicates that the pore size of the PVDF microporous filter membranes in this application is relatively large, and it is not easy to form a dense structure on the film-forming surface.

[0131] The flux of the PVDF microporous filter membranes in Examples 1 to 3 of this application is greater than that of the PVDF membranes in Comparative Examples 1 to 3, which indicates that the PVDF microporous filter membranes in this application have relatively large and uniform pore sizes.

[0132] The protein adsorption capacity of the PVDF microporous membranes in Examples 1-3 of this application is greater than that of the PVDF membranes in Comparative Examples 1-3, indicating that the PVDF microporous membranes in this application can be used for protein immunoblotting and have a better protein transfer effect.

[0133] In contrast, in Comparative Example 1, no cooling and heat preservation treatment was performed on the casting solution. Due to the high temperature of the solution, liquid-liquid phase separation and film formation occurred. As a result, the final film surface was dense, the film had a high bubble point pressure, and the flux was low.

[0134] Comparative Example 2 involves non-solvent vapor induction of a liquid membrane under high temperature and high humidity (relative conditions), which accelerates membrane phase separation, causing liquid-liquid phase separation, resulting in a relatively high bubble point pressure and low flux.

[0135] No additives were added to the casting solution of Comparative Example 3, resulting in a solution containing only polyvinylidene fluoride and solvent. DMAc is a good solvent for PVDF. Therefore, the casting solution was much more stable than that of Example 1. Consequently, when induced by low temperature and low humidity non-solvent vapor, the liquid membrane could not undergo phase separation in a short time. After the liquid membrane was placed in the coagulation bath, severe liquid-liquid phase separation occurred, resulting in a dense membrane surface, relatively high bubble point pressure, and low flux.

[0136] Furthermore, such as Figure 1 As shown, in Example 1 of this application, spherulites and rod-shaped short fiber crystals are generated on the surface of the PVDF microporous filter membrane, and there is no dense skin layer, which can be used for protein transfer and the protein transfer effect is better.

[0137] like Figure 2 As shown, in Example 2 of this application, spherulites and rod-shaped short fiber crystals are generated on the surface of the PVDF microporous filter membrane, and there is no dense skin structure. It can be used for protein transfer and the protein transfer effect is better.

[0138] like Figure 3 As shown, in Example 3 of this application, spherulites and rod-shaped short fiber crystals are generated on the surface of the PVDF microporous filter membrane, and there is no dense skin structure. It can be used for protein transfer and the protein transfer effect is better.

[0139] like Figure 4 As shown, due to the high temperature of the liquid, liquid-liquid phase separation occurs, resulting in less crystallization in the final film of Comparative Example 1. At the same time, the film surface is relatively dense, making it unsuitable for protein transfer printing.

[0140] like Figure 5 As shown, high temperature and high humidity induction will accelerate the phase separation of the membrane, causing liquid-liquid phase separation. The membrane surface of Comparative Example 2 does not form a typical crystalline spherical structure and is dense, making it unsuitable for protein transfer.

[0141] like Figure 6 As shown, the casting solution in Comparative Example 3 is stable. When steam is induced, the liquid film cannot undergo phase separation in a short time. When the liquid film is placed in the coagulation bath, a violent liquid-liquid phase separation occurs, resulting in a dense film surface, which is not suitable for protein transfer.

[0142] In summary, this invention controls the process conditions such as non-solvent vapor induction temperature and time by using appropriate raw material ratios, suitable film-forming temperatures, and simple induction methods, thereby controlling the film-forming process and preparing high-performance PVDF microporous filter membranes. These membranes are stable and can be effectively applied in the field of protein transfer with good protein transfer results.

[0143] Furthermore, such as Figure 7 As shown in the WB experiment, the PVDF microporous filter membrane of this application has a good transfer effect when performing protein transfer, with low transfer background, high sensitivity and clear bands.

[0144] Furthermore, such as Figure 8 As shown in the WB experiment, the PVDF microporous filter membrane of this application has excellent protein transfer effect on proteins of different molecular weights, with low transfer background, high sensitivity and clear bands.

[0145] Furthermore, such as Figure 9 As shown, compared with the mainstream hydrophilic modified transfer membrane 3, the transfer membrane 4 prepared by the applicant in the prior patent application has poorer sensitivity for protein adsorption.

[0146] However, as Figure 10 As shown in the WB experiment results, the PVDF microporous filter membrane 6 and membrane 7 of this application are comparable in terms of sensitivity to adsorbing target proteins, and can perform high-sensitivity detection of protein samples with microgram content.

[0147] Furthermore, this application compares the tensile strength of the aforementioned membrane 4 and the PVDF microporous filter membrane 6 of this application. The test method is as follows:

[0148] 1. Cut the membrane into 6 rectangles of 15mm*80mm each, and measure the thickness of each sample.

[0149] 2. Turn on the equipment and set the parameters according to the test requirements: gauge length 50mm, width 15mm, and thickness set according to the actual thickness of each sample.

[0150] 3. Start the test.

[0151] The test results are shown in Table 2:

[0152] Table 2 Comparison of Tensile Strength Tests

[0153] Test object Membrane 4 Tensile strength / kPa Membrane 6 Tensile strength / kPa 1 1065.01 2097.627 2 979.589 2165.633 3 960.478 2277.544 4 790.861 2097.627 5 954.954 2165.633 6 974.567 2277.544

[0154] As shown in Table 2, the PVDF microporous filter membrane 6 of this application has better tensile strength than the transfer membrane 4 prepared in the applicant's earlier patent application.

[0155] In summary, the PVDF microporous filter membrane of this application has higher sensitivity, a simpler preparation method, and lower cost compared to previous PVDF microporous filter membranes, representing a significant advancement in protein transfer applications.

[0156] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. The use of a polyvinylidene fluoride microporous filter membrane as a solid-phase carrier for protein immunoblotting, characterized in that, The surface of the polyvinylidene fluoride microporous filter membrane has spherulites and rod-shaped short fiber crystals and no dense skin layer. The uses include: the polyvinylidene fluoride microporous filter membrane is used as a solid phase carrier in the process of protein immunoblotting to perform high-sensitivity detection of protein samples with microgram content and obtain clear protein bands. The polyvinylidene fluoride microporous filter membrane is prepared by the following method: (1) At a first temperature, polyvinylidene fluoride is dissolved in a solvent and additives are added, and the mixture is mixed to obtain a liquid. The liquid is then cooled to a second temperature to obtain a casting solution. The first temperature is 40~80℃, and the second temperature is 30~60℃. The solvent includes one or more of N,N-dimethylacetamide, N,N-methylformamide, N-methyl-2-pyrrolidone, triethyl phosphate, and 2-pyrrolidone. (2) The casting solution is used to prepare a liquid film; (3) After the liquid membrane is placed in a low temperature and low humidity environment for steam-induced phase separation, it is immersed in a coagulation bath to solidify and form the polyvinylidene fluoride microporous filter membrane; the temperature of the low temperature and low humidity environment is 10~30℃, the relative humidity is 40~80RH%, and the steam induction time is 120~600s.

2. The use according to claim 1, characterized in that, In step (1), the mass fraction of polyvinylidene fluoride in the feed solution is 10~30 wt%; And / or, the solvent has a mass fraction of 50~80 wt%; And / or, the mass fraction of the additive is 0~20wt%.

3. The use according to claim 1, characterized in that, The additives include one or more of methanol, ethanol, glycerol, isopropanol, n-butanol, diethylene glycol, triethylene glycol, tetraethylene glycol, ethylene glycol methyl ether, ethylene glycol dimethyl ether, and water.

4. The use according to claim 1, characterized in that, The second temperature is 30~50℃.

5. The use according to claim 1, characterized in that, Step (3) includes: immersing in a coagulation bath to solidify and form, washing, drying, and obtaining the polyvinylidene fluoride microporous filter membrane.

6. A protein immunoblotting method, characterized in that, include: Protein samples are separated by electrophoresis and transferred on a solid-phase support, and finally detected and analyzed by antigen-antibody reaction. The solid-phase support is the polyvinylidene fluoride microporous filter membrane having the use described in any one of claims 1 to 5.

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