Application of polyvinylidene fluoride microporous filter membrane and western blotting method

Abstract

The invention belongs to the technical field of protein transfer printing, and provides application of a polyvinylidene fluoride microporous filter membrane and a protein immunoblotting method.The surface of the polyvinylidene fluoride microporous filter membrane is provided with spherocrystal and rod-like short fiber crystals and is free of a compact skin layer. According to the application, the polyvinylidene fluoride microfiltration membrane serves as a solid-phase carrier in the western blotting process to conduct high-sensitivity detection on a protein sample with the microgram-level content, and a clear protein band is obtained; the polyvinylidene fluoride microfiltration membrane is prepared by the following method: at a first temperature, dissolving polyvinylidene fluoride in a solvent, adding an additive, mixing to obtain a feed liquid, and cooling the feed liquid to a second temperature to obtain a membrane casting liquid; preparing the membrane casting solution into a liquid membrane; and carrying out steam induced phase separation on the liquid membrane, and then immersing the liquid membrane into a coagulating bath for coagulating and forming to obtain the polyvinylidene fluoride microporous filter membrane. The polyvinylidene fluoride microfiltration membrane prepared by the invention has a good protein transfer printing effect in a protein immunoblotting process, and the detection effect is particularly obviously improved.

Description

Technical Field

[0001] The present application relates to the technical field of protein transfer, and in particular to a use of a polyvinylidene fluoride microporous filter membrane and a protein immunoblotting method. Background Art

[0002] Western Blot is a technique that transfers proteins to a membrane and then detects them using antibodies. Western Blot is often used to identify a protein and can perform qualitative and semi-quantitative analysis of proteins.

[0003] Commonly used solid phase carriers in protein immunoblotting experiments include polyvinylidene fluoride (PVDF) membranes and nitrocellulose (NC) membranes. Among them, NC membranes are relatively cheap and widely used, but their binding firmness is worse than that of PVDF membranes, and their toughness is not as good as that of PVDF. They cannot be reused, but they have high protein adsorption capacity and good hydrophilicity. PVDF membranes have the advantages of higher mechanical strength, low background, wide solvent compatibility, and good staining ability, and can also be used for protein sequencing.

[0004] However, the PVDF membrane is hydrophobic and needs to be pre-wetted with anhydrous methanol to make it hydrophilic. In addition, the protein transfer bands of the PVDF membrane prepared by the prior art are not clear enough and the sensitivity is not high enough.

[0005] At present, PVDF membranes with excellent protein transfer effects are mainly manufactured abroad, which undergo hydrophilic modification of PVDF, with complex preparation processes and high costs. Domestically prepared PVDF membranes rarely achieve the same protein transfer effects as foreign membranes when used for protein immunoblotting. Summary of the invention

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

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

[0008] The first aspect of the present invention provides a use of a polyvinylidene fluoride microporous filter membrane as a solid phase carrier for protein immunoblotting, comprising: the polyvinylidene fluoride microporous filter membrane is used as a solid phase carrier in the protein immunoblotting process to perform high-sensitivity detection on a protein sample with a microgram content, and obtain clear protein bands;

[0009] The polyvinylidene fluoride microporous filter membrane is prepared by the following method:

[0010] (1) dissolving polyvinylidene fluoride in a solvent at a first temperature and adding an additive, mixing to obtain a feed liquid, and cooling the feed liquid to a second temperature to obtain a casting solution;

[0011] (2) preparing the casting liquid to form a liquid film;

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

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

[0014] In one embodiment of the present application, in the feed solution of step (1), the mass fraction of polyvinylidene fluoride is 10 to 30 wt %;

[0015] and / or, the mass fraction of the solvent is 50 to 80 wt%;

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

[0017] In one embodiment of the present 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 the present 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 the present application, step (3) includes: placing the liquid membrane in a low temperature and low humidity environment for steam-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 the present application, the steam induction time in step (3) is 120 to 600 seconds.

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

[0022] In one embodiment of the present application, step (3) includes: immersing in a coagulation bath for coagulation, washing, and drying to obtain a polyvinylidene fluoride microporous filter membrane.

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

[0024] The beneficial technical effects of the present invention are:

[0025] The present invention firstly reduces the feed liquid temperature to ensure that the feed liquid system is in a metastable state; then, under low temperature and low humidity conditions, non-solvent vapor is used to induce crystallization phase separation of the membrane to generate 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 preparing a polyvinylidene fluoride microporous filter membrane.

[0026] The polyvinylidene fluoride microporous filter membrane prepared by the present invention using a simple process can be used as a solid phase carrier to perform high-sensitivity detection of protein samples with a microgram content in a short and rapid protein immunoblotting process, and clear protein bands can be obtained. The protein transfer effect is good, and the improvement of the detection effect is particularly significant.

[0027] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings herein are incorporated into the specification and constitute a part of the specification, showing embodiments consistent with the present application, and together with the specification, are used to explain the principles of the present application. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0029] Figure 1 This is an electron microscope photograph of the surface of the PVDF microporous filter membrane of Example 1;

[0030] Figure 2 This is an electron microscope photograph of the surface of the PVDF microporous filter membrane of Example 2;

[0031] Figure 3 This is an electron microscope photograph of the surface of the PVDF microporous filter membrane of Example 3;

[0032] Figure 4 This is an electron microscope photograph of the surface of the PVDF microporous filter membrane of Comparative Example 1;

[0033] Figure 5 This is an electron microscope photograph of the surface of the PVDF microporous filter membrane of Comparative Example 2;

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

[0035] Figure 7 This is the distribution diagram of protein band markers in the GADPH transfer test of PVDF microporous filter membrane in Example 1;

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

[0037] Fig. 9 The protein band marker distribution diagram for the sensitivity test of different existing transfer membranes to the target protein.

[0038] Fig.10 This is a protein band marker distribution diagram of the sensitivity test of the PVDF microporous filter membrane and the existing transfer membrane to the target protein in Example 1. DETAILED DESCRIPTION

[0039] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as the general understanding of a person of ordinary skill in the art to which the present invention belongs. It should be understood that certain features of the present invention (described in the context of separate embodiments for clarity) may also be provided in combination in a single embodiment. On the contrary, multiple features of the present invention (described in the context of a single embodiment for brevity) may also be provided separately or in any suitable combination or in any other described embodiment of the present invention when appropriate. Certain features described in the context of various embodiments will not be considered as essential features of those embodiments unless the embodiment is inoperable without those elements. The present invention is further described below by specific specific examples, but it should be noted that the specific process conditions and results described in the embodiments of the present invention are only used to illustrate the present invention, and the protection scope of the present invention cannot be limited thereto. All equivalent changes or modifications made according to the spirit and essence of the present invention should be included in the protection scope of the present invention.

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

[0041] The present invention provides a use of a polyvinylidene fluoride microporous filter membrane as a solid phase carrier for protein immunoblotting, comprising: the polyvinylidene fluoride microporous filter membrane is used as a solid phase carrier in the protein immunoblotting process to perform high-sensitivity detection on a protein sample with a microgram content, and obtain clear protein bands;

[0042] The polyvinylidene fluoride microporous filter membrane is prepared by the following method:

[0043] (1) Dissolving polyvinylidene fluoride in a solvent at a first temperature and adding an additive, stirring and mixing the mixture to obtain a slurry, cooling the slurry to a second temperature and keeping the temperature to obtain a casting solution.

[0044] In this step, the first temperature is 40 to 80°C, preferably 50 to 70°C;

[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 to 30 wt%, preferably 15 to 25 wt%;

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

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

[0051] (2) Coating the casting liquid onto the PES release film and scraping the film to form a liquid film, with the scraping interval being 200 to 500 μm, preferably 300 to 400 μm.

[0052] (3) placing the liquid membrane in a low temperature and low humidity environment for steam-induced phase separation, wherein the temperature of the low temperature and low humidity environment is 10 to 30° C. and the relative humidity is 40 to 80 RH%.

[0053] In this step, the temperature is preferably 20 to 30°C;

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

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

[0056] (4) immersing the liquid membrane after steam-induced phase separation in a coagulation bath, exchanging the solvent and additives, coagulating into a shape, washing, and drying to obtain a polyvinylidene fluoride microporous filter membrane.

[0057] In this step, the coagulation bath temperature is 20 to 60°C, preferably 20 to 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: electrophoretically separating and transferring the protein sample on a solid phase carrier, and finally detecting and analyzing through antigen-antibody reaction, wherein the solid phase carrier is a polyvinylidene fluoride microporous filter membrane having the above-mentioned purpose.

[0063] The present invention is described in detail below by specific examples. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0064] Example 1

[0065] (1) dissolving 60 g of polyvinylidene fluoride in 180 g of N,N-dimethylacetamide, adding 60 g of ethylene glycol dimethyl ether, and stirring at 70° C. for 8 h until the polyvinylidene fluoride is uniformly dissolved to obtain a feed solution;

[0066] The material liquid was cooled to 30°C and kept warm to obtain a casting liquid.

[0067] (2) The casting liquid is coated on the PES release film and the film is scraped to form a liquid film. The scraping interval is 300 μm.

[0068] (3) The liquid film is placed in a low temperature and low humidity environment for steam-induced phase separation for 180 seconds, wherein the temperature of the low temperature and low humidity environment is 25° C. and the relative humidity is 80 RH%.

[0069] (4) immersing the liquid film after steam-induced phase separation in a coagulation bath at 25° C., wherein the coagulation bath is pure water, for 8 minutes to coagulate and form a gel film;

[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) dissolving 54 g of polyvinylidene fluoride in 216 g of N,N-dimethylacetamide, adding 27 g of ethylene glycol dimethyl ether and 3 g of water, stirring at 80° C. for 8 h until the polyvinylidene fluoride is uniformly dissolved to obtain a feed solution;

[0073] The material liquid was cooled to 40°C and kept warm to obtain a casting liquid.

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

[0075] (3) The liquid membrane is placed in a low temperature and low humidity environment for steam-induced phase separation for 240 seconds, wherein the temperature of the low temperature and low humidity environment is 20° C. and the relative humidity is 60 RH%.

[0076] (4) immersing the liquid film after steam-induced phase separation in a coagulation bath at 30° C., wherein the coagulation bath is pure water, for 5 minutes to coagulate and form a gel film;

[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) dissolving 60 g of polyvinylidene fluoride in 180 g of N,N-dimethylacetamide, adding 30 g of ethylene glycol dimethyl ether and 30 g of diethylene glycol, stirring at 70° C. for 8 h until the polyvinylidene fluoride is uniformly dissolved to obtain a feed solution;

[0080] The material liquid was cooled to 30°C and kept warm to obtain a casting liquid.

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

[0082] (3) The liquid film is placed in a low temperature and low humidity environment for steam-induced phase separation for 240 seconds, wherein the temperature of the low temperature and low humidity environment is 20° C. and the relative humidity is 70 RH%.

[0083] (4) immersing the liquid film after steam-induced phase separation in a coagulation bath at 40° C., wherein the coagulation bath is pure water, for 10 min to coagulate and form a gel film;

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

[0085] Comparative Example 1

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

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

[0088] Comparative Example 2

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

[0090] (3) The liquid film is placed in a high temperature and high humidity environment (relative conditions) for steam-induced phase separation for 180 seconds, wherein the temperature of the high temperature and high humidity environment is 35° C. and the relative humidity is 85 RH%.

[0091] Comparative Example 3

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

[0093] (1) dissolving 60 g of polyvinylidene fluoride in 180 g of N,N-dimethylacetamide, stirring at 70° C. for 8 h, until the polyvinylidene fluoride is evenly dissolved to obtain a feed solution;

[0094] The material liquid was cooled to 30°C and kept warm to obtain a casting liquid.

[0095] Performance Testing

[0096] Bubble point pressure and flux: The bubble point pressure (test liquid is anhydrous ethanol) and flux (test liquid is anhydrous ethanol) of the membranes prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were tested according to "GB / T32361-2015 Separation membrane pore size test method bubble point and average flow method". The test results are shown in Table 1.

[0097] Protein adsorption capacity: The protein adsorption capacity of the membranes prepared in Examples 1 to 3 and Comparative Examples 1 to 3 was detected by the following method:

[0098] The cross section of the film is a sample fractured in liquid nitrogen, and the surface to be observed is gold-plated by a sputtering coater in a vacuum environment. After treatment, the sample is scanned and imaged at an accelerating voltage of 10 kV. The dried transfer film is placed in a protein solution of a certain concentration and statically adsorbed for 24 hours. The change in the absorbance of the solution before and after adsorption is detected, and the amount of protein adsorbed by the film is calculated. The test results are shown in Table 1.

[0099] Microstructure: The microstructure of the membranes prepared in Examples 1 to 3 and Comparative Examples 1 to 3 was observed using an electron scanning microscope. Figures 1 to 6 shown.

[0100] Protein transfer experiment:

[0101] 1. The following experiment was conducted using the PVDF microporous 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 a uniform translucent gray;

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

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

[0106] 2. Transfer:

[0107] S1. Disperse the protein mixture on a polyacrylamide gel, wherein the target protein is GAPDH, and the protein mixture is a supernatant protein solution of HEK-293 cell disruption;

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

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

[0110] S4. Transfer proteins according to the transfer equipment manufacturer's instructions;

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

[0112] 3. Immunoassay (standard immunoassay):

[0113] S1. If the blot 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 blot in ultrapure water for 1 minute;

[0115] S3. Place the blot in blocking buffer and incubate for 1 hour with gentle agitation. Dilute the primary antibody in commercial antibody diluent, wash buffer, or blocking buffer;

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

[0117] S5. Wash with washing buffer (add Prepare commercial antibody diluent, washing or blocking buffer to dilute the secondary antibody;

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

[0119] S7. Wash the blot 3 to 5 times with wash 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 to 5 minutes according to the manufacturer's instructions.

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

[0123] The distribution diagram of protein band markers of the GADPH transfer test of the PVDF microporous filter membrane in Example 1 measured based on the above experimental method is as follows: Figure 7 shown.

[0124] 2. Using E. coli lysate as the target protein, the protein concentration gradients were 20 μg / L, 4 μg / L, and 0.8 μg / L, respectively. A protein transfer experiment was performed according to the above experimental steps to measure the protein band marker distribution diagram of the E. coli supernatant protein transfer test using the PVDF microporous filter membrane in Example 1, as shown in FIG. Figure 8 shown.

[0125] 3. Nine cell lines, including HeLa, HEK-293, Jurkat, K-562, HepG2, L02, HSC-T6, ROS1728, and NIH / 3T3, were selected, SRP9 was used as the target protein, and the transfer membrane of control group 1 and the transfer membrane of control group 2 (the transfer membrane product in the CN202311130714.X patent previously applied by the applicant) were subjected to protein transfer experiments according to the above experimental steps. The experimental results are as follows Fig. 9 shown.

[0126] 4. Nine cell lines, including U2OS, A549, HeLa, HEK-293, HepG2, K-562, HSC-T6, NIH / 3T3, and 4T1, were selected, and FNTB was used as the target protein. The PVDF microporous filter membrane of Example 1 and the transfer membrane of Control Group 1 (existing hydrophilic modified transfer membrane) were used for protein transfer experiments according to the above experimental steps. The experimental results are shown in the figure. Fig.10 shown.

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

[0128] Table 1 Properties of the films prepared in Examples 1 to 3 and Comparative Examples 1 to 3

[0129]

[0130] It can be seen from Table 1 that the bubble point pressures of the PVDF microporous membranes of Examples 1 to 3 of the present application are all lower than those of the PVDF membranes of Comparative Examples 1 to 3, which indicates that the pore size of the PVDF microporous membrane of the present application is relatively large and it is not easy to form a dense structure on its membrane-forming surface.

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

[0132] The protein adsorption amount of the PVDF microporous filter membranes of Examples 1 to 3 of the present application is greater than that of the PVDF membranes of Comparative Examples 1 to 3, which indicates that the PVDF microporous filter membranes of the present application can be used for protein immunoblotting and have a good protein transfer effect.

[0133] In the comparative example 1, when preparing the casting liquid, the material liquid is not subjected to cooling and heat preservation treatment. Due to the high temperature of the material liquid, liquid-liquid phase separation occurs to form the film. Therefore, the final film surface is dense, the membrane has a high bubble point pressure, and the flux is low.

[0134] In comparative example 2, the liquid membrane is subjected to non-solvent vapor induction in a high temperature and high humidity (relative conditions) environment, which accelerates the phase separation of the membrane, causing liquid-liquid phase separation of the membrane, and finally the bubble point pressure of the membrane is relatively high and the flux is low.

[0135] In Comparative Example 3, no additives were added to the casting liquid, resulting in only polyvinylidene fluoride and solvent in the slurry, wherein DMAc is a good solvent for PVDF. Therefore, the state of the casting liquid is very stable relative to that of the casting liquid in Example 1. Furthermore, when induced by low-temperature and low-humidity non-solvent vapor, the liquid membrane cannot undergo phase separation in a short period of time. After the liquid membrane is placed in a coagulation bath, violent liquid-liquid phase separation occurs, resulting in a dense membrane surface, a relatively high bubble point pressure, and a low flux.

[0136] Further, if Figure 1 As shown, spherulites and rod-shaped short fiber crystals are generated on the surface of the PVDF microporous filter membrane in Example 1 of the present application, and there is no dense cortex, so it can be used for protein transfer and the protein transfer effect is better.

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

[0138] like Figure 3 As shown, spherulites and rod-shaped short fiber crystals are generated on the surface of the PVDF microporous filter membrane in Example 3 of the present application, and there is no dense cortical structure, so 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 feed liquid, liquid-liquid phase separation occurred to form a film. Therefore, the final film of Comparative Example 1 had less crystals and a denser film surface, which was not suitable for protein transfer.

[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 of the membrane. The surface of the membrane of Comparative Example 2 does not form a typical crystalline spherical structure, the surface is dense, and is not suitable for protein transfer.

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

[0142] In summary, the present invention controls the process conditions such as non-solvent vapor induction temperature and time through appropriate raw material ratios, suitable film-forming temperature, and a simple induction method, so that the film-forming process is controlled, and then a PVDF microporous filter membrane with excellent performance is prepared. The performance is stable and can be effectively applied in the field of protein transfer with good protein transfer effect.

[0143] Further, if Figure 7 As shown, the WB experimental results show that the PVDF microporous filter membrane of the present application has a good transfer effect when performing protein transfer, with low transfer background, high sensitivity and clear bands.

[0144] Further, if Figure 8 As shown, the WB experimental results show that the PVDF microporous filter membrane of the present application has excellent protein transfer effect on proteins of different molecular weights, with low transfer background, high sensitivity and clear bands.

[0145] Further, if Fig. 9 As shown, compared with the hydrophilic modified transfer membrane of the mainstream competitor of membrane 3, the transfer membrane 4 prepared by the applicant in the prior patent application has poorer sensitivity to protein adsorption than membrane 3.

[0146] However, if Fig.10 As shown, the WB experimental results show that the PVDF microporous filter membrane 6 of the present application and the mainstream competitive transfer membrane 7 have comparable performance in terms of sensitivity in adsorbing target proteins, and can perform high-sensitivity detection of protein samples with microgram levels.

[0147] Furthermore, the present application conducts a tensile strength comparison test on the above membrane 4 and the PVDF microporous filter membrane 6 of the present application. The test method is as follows:

[0148] 1. Cut the film into 15mm*80mm rectangles, a total of 6 pieces, 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 testing.

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

[0152] Table 2 Comparative test of tensile strength

[0153] Test subjects Film 4 tensile strength / kPa Film 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] It can be seen from Table 2 that the PVDF microporous filter membrane 6 of the present application has better tensile strength than the transfer membrane 4 prepared in the applicant's prior patent application.

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

[0156] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A 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 cortex, and the uses include: the polyvinylidene fluoride microporous filter membrane is used as a solid phase carrier in the protein immunoblotting process 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) dissolving polyvinylidene fluoride in a solvent at a first temperature and adding an additive, mixing to obtain a feed liquid, and cooling the feed liquid to a second temperature to obtain a casting liquid; (2) preparing the casting solution to form a liquid film; (3) After the liquid membrane is subjected to steam-induced phase separation, it is immersed in a coagulation bath for coagulation and molding to obtain the polyvinylidene fluoride microporous filter membrane.

2. The use according to claim 1, characterized in that The first temperature is 40-80°C; and / or the second temperature is 30-60°C.

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

4. The use according to claim 1, characterized in that The solvent includes one or more of N,N-dimethylacetamide, N,N-methylformamide, N-methyl-2-pyrrolidone, triethyl phosphate, and 2-pyrrolidone.

5. The use according to claim 1, characterized in that: 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.

6. The use according to claim 1, characterized in that: The step (3) comprises: placing the liquid membrane in a low temperature and low humidity environment for steam-induced phase separation, wherein the temperature of the low temperature and low humidity environment is 10 to 30° C. and the relative humidity is 40 to 80 RH%.

7. The use according to claim 1, characterized in that The steam induction time in step (3) is 120 to 600 seconds.

8. The use according to claim 2, characterized in that: The second temperature is 30-50°C.

9. The use according to claim 1, characterized in that The step (3) comprises: immersing in a coagulation bath for coagulation, washing, and drying to obtain the polyvinylidene fluoride microporous filter membrane.

10. A protein immunoblotting method, characterized in that: include: The protein sample is separated by electrophoresis and transferred on a solid phase carrier, and finally detected and analyzed by antigen-antibody reaction. The solid phase carrier is the polyvinylidene fluoride microporous filter membrane having the use according to any one of claims 1 to 9.

Images