Formula and application of PVDF (Polyvinylidene Fluoride) membrane regeneration liquid

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a formula and application of a PVDF (Polyvinylidene Fluoride) membrane regeneration liquid. The formula of the PVDF membrane regeneration liquid comprises guanidine hydrochloride with the concentration of 5-6 M; the concentration of the ethyl phenyl polyethylene glycol (NP-40) is 0.2 percent to 1 percent; the concentration of the beta-mercaptoethanol is 0.05 to 0.1 M; the concentration of the trihydroxymethyl aminomethane hydrochloride (Tris-HCl) is 20 to 50 mM, and the pH value of the trihydroxymethyl aminomethane hydrochloride (Tris-HCl) is 7.5 to 8. The PVDF membrane regeneration liquid can quickly and efficiently remove primary antibodies, secondary antibodies and horse radish peroxidase compounds combined on a PVDF membrane (stripping membrane) at normal temperature, and target protein originally combined on the membrane cannot be influenced. The regenerated PVDF film can be applied to the WB experiment again, the result of the regenerated PVDF film is basically consistent with the result of the WB experiment of a new PVDF film, and the experiment time and cost are reduced on the premise that the consistency of the experiment results is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a PVDF membrane regeneration liquid formula and application. Background Art

[0002] Protein blotting (immunoblotting experiment) is also known as Western Blot (WB experiment). It is a conventional experimental method for protein analysis in molecular biology, biochemistry and immunogenetics. It is similar to the Southern Blot or Northern Blot hybridization method, but the Western Blot method uses polyacrylamide gel electrophoresis, the object to be detected is protein, the "probe" is antibody, and the "color development" uses a labeled secondary antibody. The protein sample separated by PAGE (polyacrylamide gel electrophoresis) is transferred to a solid phase carrier (such as PVDF or nitrocellulose membrane). The solid phase carrier adsorbs the protein in the form of non-covalent bonds and can keep the type of polypeptide separated by electrophoresis and its biological activity unchanged. The protein or polypeptide on the solid phase carrier is used as an antigen, and reacts with the corresponding antibody, and then reacts with the enzyme or isotope-labeled secondary antibody. After substrate color development or radioautography, the protein component expressed by the specific target gene separated by electrophoresis is detected. This technology is also widely used to detect the expression of protein levels.

[0003] There are many kinds of regeneration buffers (stripping buffer) for immunoblotting membranes on the market, and they are used in immunoblotting experiments (WB). However, the operation methods of regeneration buffers for immunoblotting membranes on the market are cumbersome and the operating conditions are limited. For quantitative experiments, there will be errors between each gel and each experiment. Secondly, many regeneration buffers for immunoblotting membranes on the market are only for nitrocellulose membranes (NC membranes) and cannot be applied to PDVF membranes. However, PVDF membranes play an important role in immunoblotting experiments (WB). Multiple targets can be detected on the same PVDF membrane, which can not only reduce experimental errors, but also save the time and cost of conventional protein immunoblotting experiments to a large extent. Therefore, the application and regeneration of PCDF membranes are crucial.

[0004] Therefore, the development of new PVDF membrane regeneration solutions and PVDF membrane regeneration methods with simple operating conditions are crucial. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the existing PVDF membrane regeneration method and provide a formula of a PVDF membrane regeneration solution and its application.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a PVDF membrane regeneration solution formulation, comprising:

[0008] Guanidine hydrochloride, concentration 5-6M;

[0009] Ethylphenyl polyethylene glycol (NP-40), concentration 0.2%-1%;

[0010] β-mercaptoethanol, concentration 0.05-0.1M;

[0011] and Tris-HCl at a concentration of 20-50 mM and a pH of 7.5-8.

[0012] In a second aspect, the present invention provides a method for regenerating a PVDF membrane, comprising the following steps:

[0013] (1) Rinse the PVDF membrane at room temperature using the PVDF membrane regeneration solution formula described in 1 above, each time for 10 minutes, and change the PVDF membrane regeneration solution three times;

[0014] (2) Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times;

[0015] (3) Development: Cover and soak the PVDF membrane with 0.5-1 ml of ECL substrate solution, and use a gel imaging system for chemiluminescence development and exposure.

[0016] In a third aspect, the present invention provides an application of the PVDF membrane regeneration solution formula as described in the first aspect, wherein the PVDF membrane regeneration solution formula is applied to immunoblotting experiments, weak binding antibody PVDF membrane regeneration experiments, strong binding antibody PVDF membrane regeneration experiments or secondary PVDF membrane regeneration experiments.

[0017] Preferably, the PVDF membrane regeneration solution formula is applied to an immunoblotting experiment, and the immunoblotting experiment comprises the following steps:

[0018] Sample processing, including preparation of lysate and extraction of tissue proteins;

[0019] SDS-PAGE gel electrophoresis;

[0020] Electroblotting and immunoblotting.

[0021] Preferably, the extraction of tissue protein comprises the following steps:

[0022] ① Use scissors and tweezers to take 30-50 mg of tissue into a 2 ml EP tube, add 1000 μl of freshly prepared lysis buffer and grind it thoroughly in a grinder;

[0023] ②Stand on ice for 30 minutes;

[0024] ③Ultrasonic disruption under ice bath conditions, and stop ultrasonication after the lysate is clarified;

[0025] ④ Centrifuge at 12000rpm, 10min, 4℃, take the supernatant as tissue protein, and store it in aliquots at -20℃ for short-term storage.

[0026] Preferably, the SDS-PAGE gel electrophoresis comprises the following steps:

[0027] ① The prepared protein sample was boiled at 100°C for 5 min and centrifuged at 12000 rpm for 1 min;

[0028] ② Transfer the glass plate with gel to the electrophoresis tank, add electrophoresis buffer, and spot the sample;

[0029] ③ Connect the power supply, start electrophoresis at 90V for 20min, and increase the current to 140V after the bromophenol blue front reaches the separation gel for 50min per gel;

[0030] ④ Stop electrophoresis when the bromophenol blue front reaches the bottom of the glass plate and mark it.

[0031] Preferably, the electrotransfer and immunoblotting comprises the following steps:

[0032] ① Before using the PVDF membrane, soak it in pure methanol for 1 minute, and then quickly soak it in the wet transfer solution;

[0033] ② Place the transfer clips, two sponge pads, filter paper and activated PVDF membrane in a basin with transfer solution;

[0034] ③Open the clamp, with the white one at the bottom and the black one at the top. Place the transfer sponge and three filter papers on the two clamps in sequence. Then place the PVDF membrane on the white side, remove the bubbles, put the cut glue on the membrane, pick up the filter paper on the black clamp and stick it on the glue on the white clamp, then stick the sponge on the black side. Finally, clamp the transfer clamp, put the black side to the black side into the transfer core, fill it with transfer solution, put the transfer tank into ice water, and transfer the membrane at a constant current of 250mA for 40-90min.

[0035] ④After the transfer, mark the marker, cut the line, and then put the membrane into the blocking solution of 5% skim milk and incubate it on a shaker at room temperature for 1 hour;

[0036] ⑤ Add 20 ml of diluent in the primary antibody incubation box, with a concentration of 0.1-5 μg / ml, cut the membrane and put it in the corresponding position of the incubation box, incubate at room temperature for 1 hour, or at 4°C overnight;

[0037] ⑥ Wash with TBST 3 times, 10 min each time;

[0038] ⑦ Add HRP enzyme-labeled secondary antibody and incubate at room temperature for 1 hour. The dilution ratio of the secondary antibody is 1:10000;

[0039] ⑧ Wash with TBST 3 times, 10 min each time;

[0040] ⑨ Development: Cover and soak the PVDF membrane with 0.5-1ml of ECL substrate solution, and develop and expose it using a gel imager.

[0041] Preferably, the PVDF membrane regeneration solution formula is applied to a weakly binding antibody PVDF membrane regeneration experiment, and the weakly binding antibody PVDF membrane regeneration experiment comprises the following steps:

[0042] (1) Immunoblotting was performed as described above, with ACTA2 as the primary antibody;

[0043] (2) PVDF membrane regeneration experiment, including:

[0044] ① Rinse the PVDF membrane at room temperature with the PVDF membrane regeneration solution formula described above, each time for 10 minutes, and change the PVDF membrane regeneration solution three times;

[0045] ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times;

[0046] ③ Development: ECL substrate solution (0.5-1 ml) covers and soaks the PVDF membrane, and the gel imager is used for chemiluminescence development and exposure.

[0047] Preferably, the PVDF membrane regeneration solution formula is applied to a strong binding antibody PVDF membrane regeneration experiment, and the strong binding antibody PVDF membrane regeneration experiment comprises the following steps:

[0048] (1) Using the immunoblotting assay described above, the primary antibody used was CKMB;

[0049] (2) PVDF membrane regeneration experiment, including:

[0050] ① Rinse the PVDF membrane at room temperature with the PVDF membrane regeneration solution formula described above, each time for 10 minutes, and change the PVDF membrane regeneration solution three times;

[0051] ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times;

[0052] ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1 ml), and use a gel imager for chemiluminescence development and exposure;

[0053] (3) Extended PVDF membrane regeneration experiment, including:

[0054] ① Rinse the PVDF membrane at room temperature with the PVDF membrane regeneration solution formula described above, each time for 15 minutes, and change the PVDF membrane regeneration solution twice;

[0055] ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times;

[0056] ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1 ml), and use a gel imager for chemiluminescence development and exposure;

[0057] (4) A regenerated membrane immunoblotting experiment was performed using the immunoblotting assay described above, with the primary antibody used being CKMB.

[0058] Preferably, the PVDF membrane regeneration solution formula is applied to a secondary PVDF membrane regeneration experiment, and the secondary PVDF membrane regeneration experiment comprises the following steps:

[0059] (1) Immunoblotting was performed as described above, with IGKC as the primary antibody;

[0060] (2) A PVDF membrane regeneration experiment, including:

[0061] ① Rinse the PVDF membrane at room temperature with the above-mentioned PVDF membrane regeneration solution for 10 minutes each time, and change the PVDF membrane regeneration solution three times;

[0062] ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times;

[0063] ③ Development: Cover and soak the PVDF membrane with 0.5-1ml of ECL substrate solution, and use a gel imager for chemiluminescence development and exposure;

[0064] (3) immunoblotting of regenerated membranes using the same immunoblotting assay as described above, with ACTA2 as the primary antibody;

[0065] (4) Secondary PVDF membrane regeneration experiment, including:

[0066] ① Rinse the PVDF membrane regenerated after the immunoblotting experiment at room temperature with the PVDF membrane regeneration solution formula described above, each time for 10 minutes, and change the PVDF membrane regeneration solution three times;

[0067] ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times;

[0068] ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1 ml), and use a gel imager for chemiluminescence development and exposure;

[0069] (5) Secondary regeneration membrane immunoblotting experiment: The immunoblotting experiment described above was performed, and the primary antibody used for incubation was ACTA2.

[0070] The applicable scope of the PVDF membrane regeneration solution as described above is applicable to targets with strong or weak binding.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] (1) The PVDF membrane regeneration solution formula provided by the present invention can not only quickly and efficiently remove the primary antibody, secondary antibody and HRP complex bound to the membrane, but also reuse the regenerated PVDF membrane to detect other target proteins.

[0073] (2) The PVDF membrane regeneration method provided by the present invention is simple and efficient. It only takes about 30 minutes at room temperature to achieve the regeneration of the protein blotting membrane, which saves time, labor, and cost. It avoids human errors caused by operations such as running gels and loading samples, and greatly saves the time and cost of conventional protein immunoblotting experiments.

[0074] (3) Using the PVDF membrane regeneration solution formula provided by the present invention, the PVDF membrane can be regenerated and regenerated repeatedly at least twice. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 The chemiluminescence results of the ACTA2 protein expression in the sample detected by immunoblotting experiment for the weakly binding ACTA2 antibody; and the chemiluminescence results after stripping the membrane using the PVDF membrane regeneration solution provided by the present invention;

[0076] Figure 2 The chemiluminescence results of the CKMB protein expression in the sample detected by immunoblotting experiments with CKMB antibodies with strong binding ability; the chemiluminescence results after conventional rinsing with PVDF membrane regeneration solution; and the chemiluminescence results after prolonged rinsing with PVDF membrane regeneration solution; and finally the chemiluminescence results after the new membrane after stripping was re-incubated with CKMB antibodies;

[0077] Figure 3 The chemiluminescence results of IGKC protein expression in samples detected by immunoblotting experiments; the chemiluminescence results after rinsing with PVDF membrane regeneration solution; the chemiluminescence results after re-incubation with ACTA2 antibody; the chemiluminescence results of secondary PVDF membrane regeneration experiments; and the chemiluminescence results after re-incubation with ACTA2 antibody after secondary stripping. DETAILED DESCRIPTION

[0078] In order to better illustrate the technical scheme of the present invention, the scheme of the present invention will be explained below in conjunction with embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If specific techniques or conditions are not indicated in the embodiments, they are carried out according to the techniques described in the literature or reference books in this area or according to the product instructions. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products.

[0079] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form a new or preferred technical solution.

[0080] The present invention aims to solve the problems of experimental errors, large operation limitations, complicated operation procedures, and limited application scope in various immunoblotting membrane regeneration solutions (stripping buffer) currently on the market. A PVDF membrane regeneration solution formula and application are provided, which has the advantages of simple operation, wide application range, strong regeneration, and reduced cost. The specific scheme is as follows:

[0081] Example 1

[0082] The first aspect of the present invention provides a PVDF membrane regeneration solution formulation, which comprises:

[0083] Guanidine hydrochloride, concentration 5-6M;

[0084] Ethylphenyl polyethylene glycol (NP-40), concentration 0.2%-1%;

[0085] β-mercaptoethanol, concentration 0.05-0.1M;

[0086] Tris-HCl, concentration 20-50 mM, pH 7.5-8.

[0087] Wherein, the concentration of guanidine hydrochloride is 5-6M; guanidine hydrochloride with a concentration of 5-6M can effectively denature proteins, destroy their secondary and tertiary structures, stretch peptide chains, and destroy the interaction between antigens and antibodies bound to the PVDF membrane, which plays an important role in the regeneration of the PVDF membrane of the present application;

[0088] Among them, ethylphenyl polyethylene glycol (NP-40), with a concentration of 0.2%-1%, is a mild non-ionic detergent. This detergent has a strong binding force with proteins and is used to prevent hydrophobic interactions between material molecules, ensure the full dissolution and structural stability of proteins, and reduce the binding of PVDF membranes to non-specific proteins to ensure that the regenerated PVDF membrane can bind to new antibodies.

[0089] Among them, β-mercaptoethanol, with a concentration of 0.05-0.1M, acts as a reducing agent to prevent the antigen bound to the membrane from being oxidized.

[0090] Among them, according to the characteristics of tris-HCl itself, when the concentration is 20-50mM, it has high solubility in water, the liquid is clear, and has a high purity of up to 99%. When the pH is 7.5-8, it is close to the physiological pH, and the performance is the most stable when used.

[0091] The PVDF membrane regeneration solution formula provided by the present invention can not only quickly and efficiently remove the primary antibody, secondary antibody and HRP complex bound to the membrane, but also can reuse the regenerated PVDF membrane to detect other target proteins.

[0092] Example 2

[0093] A second aspect of the present invention provides a method for regenerating a PVDF membrane, comprising the following steps:

[0094] (1) Rinse the PVDF membrane at room temperature with the above-mentioned PVDF membrane regeneration solution for 10 minutes each time, and change the PVDF membrane regeneration solution three times.

[0095] (2) Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times.

[0096] Specifically, the purpose of rinsing is to remove impurities and residues on the membrane, thereby restoring the performance of the membrane and extending its service life; each rinsing lasts 10 minutes and the PVDF membrane regeneration solution is replaced three times to ensure that the membrane surface is thoroughly cleaned and the chemicals or contaminants that may remain during the experiment are removed.

[0097] Specifically, 1×TBST membrane wash solution contains Tween-20, a non-ionic detergent that can effectively destroy nonspecific binding on the membrane while maintaining the activity of the protein. The 1×TBST membrane wash solution is changed three times for 5 minutes each time to remove unbound antibodies and other impurities, ensure that the background signal is washed away, and make the signal of the target protein clearer, thereby improving the clarity and accuracy of the experimental results.

[0098] The PVDF membrane regeneration method provided by the present invention is simple and efficient. It only takes about 30 minutes at room temperature to achieve the regeneration of the protein blotting membrane, which saves time, labor and cost, avoids human errors caused by operations such as running gels and loading samples, and saves time and cost of conventional protein immunoblotting experiments to a great extent.

[0099] Example 3

[0100] The third aspect of the present invention provides an application of the PVDF membrane regeneration solution formula as described above. Using the PVDF membrane regeneration solution formula provided by the present invention, the PVDF membrane can be regenerated and regenerated repeatedly at least twice. Specifically, the following applications are included:

[0101] Application 1: WB experiment

[0102] The WB experimental steps are as follows:

[0103] (1) Sample processing

[0104] A. Preparation of lysis solution: Taking the mass or volume of 100 ml lysis solution as an example, the formula is shown in Table 1:

[0105] Table 1

[0106]

[0107] B. Extraction of tissue proteins

[0108] ① Use scissors and tweezers to take 30-50 mg of tissue into a 2 ml EP tube, add 1000 μl of freshly prepared lysis buffer and grind it thoroughly in a grinder.

[0109] ② Leave on ice for 30 minutes.

[0110] ③Ultrasonicate the cells under ice bath conditions and stop sonicating when the lysate is relatively clear.

[0111] ④ Centrifuge at 12000rpm, 10min, 4℃. The supernatant is tissue protein. Aliquot and store at -20℃ for short term.

[0112] (2) SDS-PAGE gel electrophoresis

[0113] ① The prepared protein sample was boiled at 100℃ for 5 min and centrifuged at 12000 rpm for 1 min.

[0114] ② Transfer the glass plate with gel to the electrophoresis tank, add electrophoresis buffer, and apply sample (10-15μl for 1mm thick gel).

[0115] ③ Connect the power supply and start electrophoresis at 90V for about 20 minutes. After the front of bromophenol blue reaches the separation gel, increase the current to 140V and run each gel for about 50 minutes.

[0116] ④ Stop electrophoresis when the front of bromophenol blue reaches the bottom of the glass plate and mark it.

[0117] (3) Electrotransfer and immunoblotting

[0118] ①Before using the PVDF membrane, it needs to be soaked in pure methanol for 1 minute and then quickly immersed in the wet transfer solution.

[0119] ② Place the transfer clip, two sponge pads, filter paper and activated PVDF membrane in a basin with transfer solution. Pay attention to the positive and negative poles of the transfer clip.

[0120] ③Open the clamps, white on the bottom, black on the top. Place the transfer sponge and 3 filter papers on the clamps on both sides in turn, then put a PVDF membrane of appropriate size on the white side, remove the bubbles, gently put the cut glue on the membrane, be careful not to generate bubbles, pick up the filter paper on the black side of the clamp, gently stick it on the glue on the white side of the clamp, then stick the sponge on the black side, and finally gently clamp the transfer clamp, put the black side to the black side into the transfer core, fill it with transfer liquid, prepare a basin of ice water, put the transfer tank into the ice water, and transfer the membrane at a constant current of 250mA for 40-90min. If there are large molecules, increase the transfer time appropriately, and if there are small molecules, reduce the transfer time appropriately.

[0121] ④ After the transfer is completed, mark the marker, cut the line, and then place the membrane in the blocking solution (5% skim milk) and incubate on a shaker at room temperature for 1 hour.

[0122] ⑤ Add 20 ml of diluent to the primary antibody incubation box in sequence, with a recommended concentration of 0.1-5 μg / ml, cut the membrane and place it in the corresponding position of the incubation box, incubate at room temperature for 1 hour or at 4°C overnight.

[0123] ⑥ Wash three times with TBST, 10 min each time.

[0124] ⑦Add HRP enzyme-labeled secondary antibody and incubate at room temperature for 1 hour. The dilution ratio of the secondary antibody is 1:10000.

[0125] ⑧Wash three times with TBST, 10 min each time.

[0126] ⑨ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1 ml), and develop and expose it using a gel imager.

[0127] Application 2: Weakly Binding Antibody PVDF Membrane Regeneration Experiment

[0128] The specific steps include:

[0129] (1) WB experiment

[0130] The steps are the same as above, and the specific primary antibody used for incubation is ACTA2 (from Biolight)

[0131] (2) PVDF membrane regeneration experiment

[0132] ① Rinse the PVDF membrane with the above-mentioned PVDF membrane regeneration solution at room temperature for 10 minutes each time, and change the PVDF membrane regeneration solution three times. ② Rinse the PVDF membrane in 1×TBST membrane washing solution for 5 minutes each time, and change the 1×TBST membrane washing solution three times. ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1ml), and perform chemiluminescence development exposure with a gel imaging instrument.

[0133] like Figure 1 As shown, (a) is the chemiluminescence result of detecting the expression of ACTA2 protein in the sample by immunoblotting experiment with an ACTA2 antibody with weaker binding ability; (b) is the chemiluminescence result after stripping the PVDF membrane using the regeneration solution provided by the present invention; the samples in each lane are: mouse heart, mouse spleen, mouse lung, mouse brain, mouse heart, and mouse spleen.

[0134] Depend on Figure 1 The results showed that no bands of the target protein appeared after the development exposure after regeneration, indicating that the PVDF membrane regeneration method provided by the present invention can remove the primary antibody, secondary antibody and HRP complex for weakly bound antibodies by rinsing with PVDF membrane regeneration solution at room temperature for 30 minutes.

[0135] Application 3: Strong binding antibody PVDF membrane regeneration experiment

[0136] The specific steps include:

[0137] (1) WB experiment

[0138] The steps were the same as above, and the specific primary antibody used for incubation was CKMB (from Biolight).

[0139] (2) PVDF membrane regeneration experiment

[0140] ① Rinse the PVDF membrane with the above-mentioned PVDF membrane regeneration solution at room temperature for 10 minutes each time, and change the PVDF membrane regeneration solution three times. ② Rinse the PVDF membrane in 1×TBST membrane washing solution for 5 minutes each time, and change the 1×TBST membrane washing solution three times. ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1ml), and perform chemiluminescence development exposure with a gel imaging instrument.

[0141] (3) Prolonged PVDF membrane regeneration experiment

[0142] ① Rinse the PVDF membrane with the above-mentioned PVDF membrane regeneration solution at room temperature for 15 minutes each time, and change the PVDF membrane regeneration solution twice. ② Rinse the PVDF membrane in 1×TBST membrane washing solution for 5 minutes each time, and change the 1×TBST membrane washing solution three times. ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1ml), and perform chemiluminescence development exposure with a gel imaging instrument.

[0143] (4) Primary regeneration membrane WB experiment

[0144] The steps were the same as above, and the specific primary antibody used for incubation was CKMB (from Biolight).

[0145] like Figure 2 As shown, (a) shows the chemiluminescence results of the CKMB protein expression of the sample detected by immunoblotting experiment with a CKMB antibody with strong binding ability; (b) shows the chemiluminescence results after conventional rinsing with PVDF membrane regeneration solution; (c) shows the chemiluminescence results after prolonged rinsing with PVDF membrane regeneration solution; (d) shows the chemiluminescence results after the new membrane after stripping is re-incubated with CKMB antibody. Among them, the samples in each lane are: human heart, human skeletal muscle, mouse heart, mouse skeletal muscle, CKMB recombinant protein.

[0146] according to Figure 2 The results show that after conventional 30-minute rinsing, some strongly bound antibodies, such as the CKMB antibody in this example, need to be rinsed with PVDF membrane regeneration solution for a longer time before they can be removed. And the regenerated membrane can still be used for WB experiments, and the experimental results are consistent with those of the new membrane.

[0147] Application 4: Secondary PVDF membrane regeneration experiment

[0148] The specific steps include:

[0149] (1) WB experiment

[0150] The steps were the same as above, and the specific primary antibody used for incubation was IGKC (from Biolight).

[0151] (2) A PVDF membrane regeneration experiment

[0152] ① Rinse the PVDF membrane with the above-mentioned PVDF membrane regeneration solution at room temperature for 10 minutes each time, and change the PVDF membrane regeneration solution three times. ② Rinse the PVDF membrane in 1×TBST membrane washing solution for 5 minutes each time, and change the 1×TBST membrane washing solution three times. ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1ml), and perform chemiluminescence development exposure with a gel imaging instrument.

[0153] (3) One-time regeneration membrane WB experiment

[0154] The steps were the same as above, and the specific primary antibody used for incubation was ACTA2 (from Biolight).

[0155] (4) Secondary PVDF membrane regeneration experiment

[0156] ① Rinse the PVDF membrane after WB experiment with the above PVDF membrane regeneration solution at room temperature for 10 minutes each time, and change the PVDF membrane regeneration solution three times. ② Rinse the PVDF membrane in 1×TBST membrane washing solution for 5 minutes each time, and change the 1×TBST membrane washing solution three times. ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1ml), and perform chemiluminescence development exposure with a gel imager.

[0157] (5) Secondary regeneration membrane WB experiment

[0158] The steps were the same as above, and the specific primary antibody used for incubation was ACTA2 (from Biolight).

[0159] like Figure 3 As shown, (a) shows the chemiluminescence results of the sample IGKC protein expression detected by immunoblotting experiment; (b) shows the chemiluminescence results after rinsing with PVDF membrane regeneration solution; (c) shows the chemiluminescence results after re-incubation with ACTA2 antibody; (d) shows the chemiluminescence results of the secondary PVDF membrane regeneration experiment; (e) shows the chemiluminescence results after re-incubation with ACTA2 antibody after secondary membrane stripping. Among them, the samples in each lane are: mouse heart, mouse spleen, mouse lung, mouse brain, mouse heart, mouse spleen.

[0160] according to Figure 3 The results show that the PVDF membrane regeneration method provided by the present invention can be repeated at least twice, and the WB test results of the two regenerated membranes are consistent with the results of the new membrane.

[0161] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A PVDF membrane regeneration solution formula, characterized in that: include: Guanidine hydrochloride, concentration 5-6M; Ethylphenyl polyethylene glycol (NP-40), concentration 0.2%-1%; β-mercaptoethanol, concentration 0.05-0.1M; and Tris-HCl at a concentration of 20-50 mM and a pH of 7.5-8.

2. A method for regenerating a PVDF membrane, characterized in that The steps include: (1) Rinse the PVDF membrane at room temperature using the PVDF membrane regeneration solution formula as described in claim 1, each time for 10 minutes, and change the PVDF membrane regeneration solution three times; (2) Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times; (3) Development: Cover and soak the PVDF membrane with 0.5-1 ml of ECL substrate solution, and use a gel imaging system for chemiluminescence development and exposure.

3. An application of the PVDF membrane regeneration solution formula as claimed in claim 1, characterized in that: The PVDF membrane regeneration liquid formula is applied to immunoblotting experiments, weakly bound antibody PVDF membrane regeneration experiments, strongly bound antibody PVDF membrane regeneration experiments or secondary PVDF membrane regeneration experiments.

4. The use according to claim 3, characterized in that: The PVDF membrane regeneration solution formula is applied to an immunoblotting experiment, and the immunoblotting experiment comprises the following steps: Sample processing, including preparation of lysate and extraction of tissue proteins; SDS-PAGE gel electrophoresis; Electroblotting and immunoblotting.

5. The use according to claim 4, characterized in that: The extraction of tissue protein comprises the following steps: ① Use scissors and tweezers to take 30-50 mg of tissue into a 2 ml EP tube, add 1000 μl of freshly prepared lysis buffer and grind it thoroughly in a grinder; ②Stand on ice for 30 minutes; ③Ultrasonic disruption under ice bath conditions, and stop ultrasonication after the lysate is clarified; ④ Centrifuge at 12000rpm, 10min, 4℃, take the supernatant as tissue protein, and store it in aliquots at -20℃ for short-term storage.

6. The use according to claim 4, characterized in that: The SDS-PAGE gel electrophoresis comprises the following steps: ① The prepared protein sample was boiled at 100°C for 5 min and centrifuged at 12000 rpm for 1 min; ② Transfer the glass plate with gel to the electrophoresis tank, add electrophoresis buffer, and spot the sample; ③ Connect the power supply, start electrophoresis at 90V for 20min, and increase the current to 140V after the bromophenol blue front reaches the separation gel for 50min per gel; ④ Stop electrophoresis when the bromophenol blue front reaches the bottom of the glass plate and mark it.

7. The use according to claim 4, characterized in that: The electro-transfer and immunoblotting process comprises the following steps: ① Before using the PVDF membrane, soak it in pure methanol for 1 minute, and then quickly soak it in the wet transfer solution; ② Place the transfer clips, two sponge pads, filter paper and activated PVDF membrane in a basin with transfer solution; ③Open the clamp, with the white one at the bottom and the black one at the top. Place the transfer sponge and three filter papers on the two clamps in sequence. Then place the PVDF membrane on the white side, remove the bubbles, put the cut glue on the membrane, pick up the filter paper on the black clamp and stick it on the glue on the white clamp, then stick the sponge on the black side. Finally, clamp the transfer clamp, put the black side to the black side into the transfer core, fill it with transfer solution, put the transfer tank into ice water, and transfer the membrane at a constant current of 250mA for 40-90min. ④After the transfer, mark the marker, cut the line, and then put the membrane into the blocking solution of 5% skim milk and incubate it on a shaker at room temperature for 1 hour; ⑤ Add 20 ml of diluent in the primary antibody incubation box, with a concentration of 0.1-5 μg / ml, cut the membrane and put it in the corresponding position of the incubation box, incubate at room temperature for 1 hour, or at 4°C overnight; ⑥ Wash with TBST 3 times, 10 min each time; ⑦ Add HRP enzyme-labeled secondary antibody and incubate at room temperature for 1 hour. The dilution ratio of the secondary antibody is 1:10000; ⑧ Wash with TBST 3 times, 10 min each time; ⑨ Development: Cover and soak the PVDF membrane with 0.5-1ml of ECL substrate solution, and develop and expose it using a gel imager.

8. The use according to claim 3, characterized in that: The PVDF membrane regeneration solution formula is applied to a weak binding antibody PVDF membrane regeneration experiment, and the weak binding antibody PVDF membrane regeneration experiment comprises the following steps: (1) using the immunoblotting assay as described in claim 4, wherein the primary antibody used in the incubation is ACTA2; (2) PVDF membrane regeneration experiment, including: ① Rinse the PVDF membrane at room temperature using the PVDF membrane regeneration solution formula as described in claim 1, each time for 10 minutes, and change the PVDF membrane regeneration solution three times; ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times; ③ Development: ECL substrate solution (0.5-1 ml) covers and soaks the PVDF membrane, and the gel imager is used for chemiluminescence development and exposure.

9. The use according to claim 3, characterized in that: The PVDF membrane regeneration solution formula is applied to a strong binding antibody PVDF membrane regeneration experiment, and the strong binding antibody PVDF membrane regeneration experiment comprises the following steps: (1) using the immunoblotting assay as described in claim 4, wherein the primary antibody used in the incubation is CKMB; (2) PVDF membrane regeneration experiment, including: ① Rinse the PVDF membrane at room temperature using the PVDF membrane regeneration solution formula as described in claim 1, each time for 10 minutes, and change the PVDF membrane regeneration solution three times; ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times; ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1 ml), and use a gel imager for chemiluminescence development and exposure; (3) Extended PVDF membrane regeneration experiment, including: ① Rinse the PVDF membrane at room temperature using the PVDF membrane regeneration solution formula as described in claim 1, each time for 15 minutes, and change the PVDF membrane regeneration solution twice; ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times; ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1 ml), and use a gel imager for chemiluminescence development and exposure; (4) A regenerated membrane immunoblotting experiment, using the immunoblotting experiment as described in claim 4, and the primary antibody used for incubation is CKMB.

10. The use according to claim 3, characterized in that: The PVDF membrane regeneration solution formula is applied to a secondary PVDF membrane regeneration experiment, and the secondary PVDF membrane regeneration experiment comprises the following steps: (1) using the immunoblotting assay as described in claim 4, wherein the primary antibody used in the incubation is IGKC; (2) A PVDF membrane regeneration experiment, including: ① Rinse the PVDF membrane at room temperature with the above-mentioned PVDF membrane regeneration solution for 10 minutes each time, and change the PVDF membrane regeneration solution three times; ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times; ③ Development: Cover and soak the PVDF membrane with 0.5-1ml of ECL substrate solution, and use a gel imager for chemiluminescence development and exposure; (3) a regenerated membrane immunoblotting experiment, using the immunoblotting experiment as described in claim 4, and the primary antibody used for incubation is ACTA2; (4) Secondary PVDF membrane regeneration experiment, including: ① Rinse the PVDF membrane regenerated after the immunoblotting experiment at room temperature with the PVDF membrane regeneration solution formula as described in claim 1, each time for 10 minutes, and change the PVDF membrane regeneration solution three times; ② Rinse the PVDF membrane in 1×TBST washing solution for 5 minutes each time, changing the 1×TBST washing solution three times; ③ Development: Cover and soak the PVDF membrane with ECL substrate solution (0.5-1 ml), and use a gel imager for chemiluminescence development and exposure; (5) Secondary regeneration membrane immunoblotting experiment, using the immunoblotting experiment as described in claim 4, and the primary antibody used for incubation is ACTA2.

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