Protein immunoblotting membrane regeneration solution and use method thereof

Abstract

The present invention relates to the field of antibody detection, and specifically to a protein immunoblot membrane regeneration solution and a use method thereof. The protein immunoblot membrane regeneration solution comprises crotonic acid in a formula with a concentration of 5-20% by mass. Horseradish peroxidase (HRP) bound to a secondary antibody is inactivated by the crotonic acid, thereby enabling reuse of the protein immunoblot membrane. The regeneration solution and the use method thereof do not affect the binding between the antibody and the antigen on the blot membrane, do not cause the loss of the original protein on the blot membrane, and are reusable. Furthermore, the regeneration solution has unique advantages in the treatment of NC blot membranes: pre-incubation with CA can not only stabilize the NC blot membrane-bound protein, but also improve the sensitivity of NC membrane analysis.

Description

Technical Field

[0001] The present invention relates to the field of antibody detection, in particular to a protein immunoblotting membrane regeneration solution and a use method thereof. Background Art

[0002] Since its introduction in 1979, western blotting (WB) has become a simple and inexpensive technique. Currently, WB is widely used for protein detection and semi-quantification, as well as for studying protein expression levels. The method essentially involves binding a specific primary antibody to the antigen on the blot membrane, followed by binding to a horseradish peroxidase (HRP)-conjugated secondary antibody. Finally, the antibody reacts with the HRP substrate, luminol, which then releases the signal as light. In practice, repeated stripping and re-probing are often performed to detect different proteins on the blot to save time and money. However, each round of stripping removes some of the proteins transferred to the blot, affecting experimental results. Therefore, it is important to determine whether HRP activity can be inactivated, allowing blots to be reused without affecting antibody binding to the antigen on the blot or the amount of protein present on the blot. To date, endogenous HRP activity has been commonly inhibited in immunohistochemistry, including by pretreatment with hydrogen peroxide, sodium azide, and phenylhydrazine. Meanwhile, four methods for HRP inactivation have been reported for Western blotting, including H2O2, sodium azide, 3,3-diaminobenzidine, or Vector SG and acetic acid. Each method has its own advantages. Nevertheless, we aimed to investigate whether there are other suitable chemicals that can regenerate the blot for continuous chemiluminescence Western blotting through HRP inhibition studies. Summary of the Invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a new protein immunoblotting membrane regeneration solution and a method for using the same.

[0004] The technical solution adopted by the present invention is as follows: a protein immunoblotting membrane regeneration solution, the regeneration solution formula includes crotonic acid, and preferably, the crotonic acid concentration is 5%-20% by mass.

[0005] Preferably, the crotonic acid concentration is 10%-20% by mass.

[0006] Also provided is a method for using a protein immunoblotting membrane regeneration solution, comprising the following steps:

[0007] Step (1): using a protein immunoblotting membrane, horseradish peroxidase, and chemiluminescence reagent to perform protein immunoblotting detection;

[0008] Step (2): Washing off the chemiluminescent reagent on the protein immunoblotting membrane used in step (1);

[0009] Step (3): adding the protein immunoblot membrane from which the chemiluminescent reagent has been washed in step (2) to the protein immunoblot membrane regeneration solution according to any one of claims 1 to 3 for shaking and incubation;

[0010] Step (4): remove the membrane in step (2), wash with double distilled water and / or TBST solution to obtain a membrane in which horseradish peroxidase has been inactivated;

[0011] Step (5): The membrane in which horseradish peroxidase has been inactivated is obtained by step (4), and protein immunoblotting is performed using horseradish peroxidase and chemiluminescence reagent.

[0012] Preferably, the protein detected in step (5) is not the same protein as the protein detected in step (1).

[0013] Preferably, steps (1) to (4) are repeated at least twice, and the protein detected later is a non-isotype protein from the protein detected earlier.

[0014] Preferably, the solid phase support of the protein immunoblotting membrane is a polyvinylidene fluoride membrane or a nitrocellulose membrane, wherein the nitrocellulose membrane is the most preferred.

[0015] The beneficial effects of the present invention are as follows: the formula of the protein immunoblotting membrane regeneration solution includes crotonic acid, and the concentration of the crotonic acid is 5-20% by mass. The protein immunoblotting membrane is reused by inactivating horseradish peroxidase (HRP) labeled with a secondary antibody with crotonic acid. The regeneration solution and the method for using the protein immunoblotting membrane regeneration solution do not affect the binding between the antibody and the antigen on the blot and the change in the antigen content on the blot membrane, and can be reused. It also has unique advantages in the treatment of NC membranes: pre-incubation with CA can not only stabilize the NC membrane-bound protein, but also improve the sensitivity of NC membrane detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying any creative work, other drawings obtained based on these drawings still fall within the scope of the present invention.

[0017] Figure 1:Dot blot hybridization and western blotting were used to evaluate the inactivation effect of different concentrations of crotonic acid (CA) on horseradish peroxidase (HRP);

[0018] A) Anti-rabbit IgG-HRP was spotted at a concentration of 80 ng / μL onto NC and PVDF membranes and treated under the indicated conditions. After the addition of CA, the blots were placed in a sealed plastic container in an incubator (40°C) for 30 minutes. After a 5-minute wash with double-distilled water, each blot was exposed to an ECL substrate and imaged simultaneously. B) Supernatants from mouse cortical protein lysates were subjected to two Western blotting (WB). The first was probed for protein kinase B (Akt). Subsequently, the NC blots were incubated with 5% CA (B), 10% CA (C), and 20% CA (D) at 40°C for 30 minutes, washed with double-distilled water for 5 minutes, exposed to a highly sensitive chemiluminescent substrate, and developed. E) Western blotting was performed using the same samples and procedures as in (B). The first was probed for phospho-p38 MAPK (p-p38 MAPK). The NC blot was then incubated with 20% CA, exposed to ECL substrate, and developed. “*” indicates a red labeled band of approximately 70 kDa.

[0019] Figure 2 Comparison of the binding capacity of proteins of varying molecular weight to NC membranes and PVDF membranes after treatment with double-distilled water and 20% CA. A) Supernatant from mouse cortical lysate was loaded in triplicate for Western blotting (WB) (15 µg / lane; three replicate lanes). Immediately after protein transfer to NC membranes, the NC membranes were soaked in double-distilled water or 20% CA at 40°C for 30 minutes. After a 30-minute soak, the membranes were washed with double-distilled water and TBST, blocked with milk, and probed with an HRP-conjugated secondary antibody. B) The same treatment steps as in (A) were used, except that the NC membrane was replaced with a PVDF membrane. "&" indicates a band approximately 17 kDa.

[0020] Figure 3In consecutive Western blotting (WB), 20% CA inactivated HRP on the NC membrane, allowing detection of phosphorylated and non-phosphorylated proteins. A-B) The same lysate from mouse cortex was used for Western blotting (15 µg / lane). Immediately after transfer, the blot was pre-incubated with double-distilled water (40°C, 30 minutes) and then blocked with milk. Following the initial Atg7 detection shown in panels A and B, the NC membrane was again soaked in 20% CA (40°C, 30 minutes). After a 30-minute soak, the blot was washed with double-distilled water and TBST and reprobed with antibodies against Atg7 (A) and phospho-JNK (p-JNK) (B). CD) The blot was preincubated with 20% CA (40°C, 30 minutes) and blocked in milk. The blot was then probed for Atg7 as shown in panels C and D. Next, the blot was soaked again in 20% CA (40°C, 30 minutes) and reprobed for Atg7 (C) and p-JNK (D). E) The same samples and processing steps as in (AB) were used for Western blotting, initially probing for p-JNK, followed by treatment with 20% CA and re-exposure to a more sensitive chemiluminescent substrate. The blot was then reprobed for total JNK. F) After the first round of 20% CA treatment, the blot was probed for p-JNK, then HRP was inactivated by a second 20% CA treatment, re-imaged, and finally reprobed for total JNK. "*" indicates a red-labeled band of approximately 70 kDa.

[0021] Figure 4 Three rounds of HRP inactivation in 20% CA in sequential Western blotting. Immediately after electrotransfer of mouse cortical lysates, the NC blot was preincubated with double-distilled water or 20% CA (40°C, 30 minutes). The blot was then probed with anti-p-p38 MAPK (A). The same NC membrane as in (A) was inactivated with 20% CA for the first time and then probed for Atg7 in the second round (B). The same NC membrane as in (B) was inactivated with 20% CA for the second time and then probed for caspase 3 in the third round (C). The same NC membrane as in (C) was inactivated with 20% CA for the third time and then probed for vinculin in the fourth round (D). "#" and solid triangles indicate residual bands of p-p38 MAPK and caspase 3, respectively. "*" indicates a red-labeled band of approximately 70 kDa. DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0023] 1. Materials and Methods

[0024] Crotonic acid ([113018], Sigma Aldrich, Taufkirchen, Germany);

[0025] 0.45 μm pore size NC membrane and PVDF membrane (Millipore, Massachusetts, United States); 0.2 μm pore size NC membrane (PALL, United States);

[0026] BeyoColor pre-stained protein markers;

[0027] BeyoECL Star chemiluminescent substrate;

[0028] Bradford protein assay kit;

[0029] RIPA buffer (Beyotime Biotechnology, Shanghai, China);

[0030] 3-color protein loading marker (TOROIVD, Shanghai, China);

[0031] Constant temperature incubator oscillator (THZ-103B, Shanghai, China);

[0032] 4-week-old, 23-g adult male C57BL / 6J mice, obtained from Gem Pharma tech Co., Ltd (manufactured under license number: SCXK-(SU)-2018-0008; Nanjing, China);

[0033] This study received ethics approval from the Wenzhou Medical University Medical Ethics Committee. All experimental procedures were approved by the Wenzhou Medical University Animal Care and Use Committee (No. 2019-75). Mice were maintained at 24–25°C on a 12 / 12 hour day / night cycle and provided with access to water and food.

[0034]

[0035] 2. Abbreviations

[0036] Crotonic acid (CA); room temperature (RT); horseradish peroxidase (HRP); phosphorylated-JNK (p-JNK); phosphorylated-mitogen-activated protein kinase p38 antibody (p-p38 MAPK); primary antibody; polyvinylidene fluoride (PVDF); nitrocellulose membrane (NC membrane).

[0037] 3. Implementation Methods

[0038] 3.1 Dot blot hybridization assay and crotonic acid treatment

[0039] The testing procedure is carried out according to the existing processing flow [1] , with minor modifications. The modifications are as follows: HRP-conjugated goat anti-rabbit IgG was diluted at a concentration of 80 ng / μL in 5% skim milk in buffered saline (0.1% Tween 20 (TBST)). The NC membrane was then spotted with 1 μL of the diluted antibody-HRP and air-dried at room temperature for 30 minutes before treatment with crotonic acid. Because PVDF membranes are naturally hydrophobic, they were soaked in methanol for 3 minutes and then rinsed with double-distilled water for a few seconds. Next, they were placed in 0.1 M (mol / L), pH 7.4 phosphate-buffered saline (PBS) for 10 minutes. Finally, the PVDF membrane was removed and air-dried for experimental use.

[0040] Because crotonic acid (CA) solidifies into solid crotonic acid crystals at room temperature, all protein immunoblot membranes were incubated with double-distilled water and crotonic acid (5% by weight, pH = 1.80; 10% by weight, pH = 1.63; 20% by weight, pH = 1.54) at 40°C for a period of time. At 40°C, all protein immunoblot membranes in the same experiment were processed in a closed container on a shaker set at 40°C (protein side down). ) After CA treatment, the membrane was immediately washed in double-distilled water for 5 minutes and then immersed in double-distilled water until it was removed and the enzyme substrate was added for detection (protein side up).

[0041] 3.2WB Analysis

[0042] WB is carried out according to existing means [2][3]Mouse cortex was removed and cut into small pieces. The fragments were incubated on ice in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, leupeptin, pH 7.4; Beyotime Biotechnology, [P0013B]). The fragments were then dispersed using ultrasound (VOSHIN-650W; Voshin, Wuxi, China) until the lysate became clear. The cortical lysate was centrifuged at 13,000 g for 30 minutes at 4°C, and the supernatant was collected. Total protein concentration was determined using a protein detection kit (P0006C; Beyotime Biotechnology). Proteins (15 μg) were separated by electrophoresis on a Tris-glycine SDS-polyacrylamide gel and transferred to a NC or PVDF membrane. After incubating the membrane in 5% skim milk in TBST for 1 hour at room temperature, the membrane was washed with TBST and incubated with primary antibodies (see Table 1) overnight at 4°C. The NC or PVDF membrane was then washed with TBST and incubated with HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG secondary antibodies in TBST for 1 hour (see Table 1). Protein signals were visualized using BeyoECL Star enhanced chemiluminescence reagents. Finally, images were acquired at a quality of at least 600 dpi using an Amersham Imager 680 (GE Healthcare Biosciences, Uppsala, Sweden). Prior to re-detection, the membrane was washed with double-distilled water for 5 minutes to remove the chemiluminescent substrate and treated with various concentrations of CA solution. Finally, all procedures were performed under identical conditions for parallel comparison.

[0043] 4. Effect of Crotonic Acid on Horseradish Peroxidase (HRP) Activity

[0044] To investigate whether different concentrations of CA affect the inactivation of HRP on protein immunoblotting membranes, parallel experiments were conducted using Comparative Example 1 and Examples 1-3. To investigate whether CA-treated protein immunoblotting membranes would cause protein loss or lead to primary antibody misrecognition, we designed Test Examples 1-2.

[0045] Comparative Example 1

[0046] 1 μL of 80 ng / μL HRP-conjugated goat anti-rabbit IgG dilution was taken and spotted on NC and PVDF membranes. After the protein immunoblotting membrane was air-dried at room temperature, it was incubated with double-distilled water at 40°C for 30 min.

[0047] Example 1

[0048] 1 μL of 80 ng / μL HRP-conjugated goat anti-rabbit IgG was spotted on NC and PVDF membranes. After the protein immunoblotting membrane was air-dried at room temperature, it was incubated with 5% CA at 40°C for 30 min.

[0049] Example 2

[0050] 1 μL of 80 ng / μL HRP-conjugated goat anti-rabbit IgG dilution was taken and spotted on NC and PVDF membranes. After the protein immunoblotting membrane was air-dried at room temperature, it was incubated with 10% CA at 40°C for 30 min.

[0051] Example 3

[0052] 1 μL of 80 ng / μL HRP-conjugated goat anti-rabbit IgG dilution was taken and spotted on NC and PVDF membranes. After the protein immunoblotting membrane was air-dried at room temperature, it was incubated with 20% CA at 40°C for 30 min.

[0053] Test Example 1

[0054] CA-treated NC blots were tested with five primary antibodies against proteins with different molecular weights, and double-distilled water-treated NC membranes were used as controls. The treatment conditions were incubated at 40°C for 30 minutes. The five primary antibodies were: Vinculin protein (116KDa; high molecular weight protein), Akt, p-Akt, p-ERK (56KDa, 60KDa and 42 / 44KDa; medium molecular weight proteins) and casapse 3 (17 / 35KDa; low molecular weight protein).

[0055] Test Example 2

[0056] The difference between Test Example 2 and Test Example 1 is that the NC imprinted membrane is replaced with a PVDF imprinted membrane.

[0057] 4.1 Results Analysis

[0058] Compared with double distilled water treatment, 5% CA treatment had no significant effect on the chemiluminescence signal of NC and PVDF blot membranes. However, 10% CA and 20% CA treatment had a particularly significant effect on NC and PVDF blot membranes, with almost no chemiluminescence signal observed ( Figure 1A). Furthermore, the background of NC blots treated with 10% or 20% CA appeared to be clearer than that of PVDF blots treated with CA. Therefore, we investigated the effect of CA treatment on NC blots and found that 20% CA treatment was more effective than 5% and 10% CA treatments when detecting protein kinase B (Akt; 56 kDa) in the blots, with a clear background ( Figure 1 BD). In addition, after the NC blot membrane was treated with 20% CA, no original signal of p-p38 MAPK was observed, and the background of the protein immunoblot membrane was also clear ( Figure 1 E). Therefore, the experimental results confirmed that horseradish peroxidase can indeed be inactivated by a certain concentration of CA.

[0059] The results of test example 1 showed that pre-incubation with 20% CA improved the detection sensitivity of casapse 3 and p-Akt compared with pre-incubation of NC blot membrane with double distilled water. However, no significant signal difference was observed for Vinculin, Akt and p-ERK ( Figure 2 A). Meanwhile, the 17 kDa casapse 3 isoform (marked by &) was present in the CA-treated group, but its band signal was very weak in the double-distilled water-treated group, indicating that pre-incubation with CA can immobilize small molecular weight proteins on the NC membrane.

[0060] In Test Example 2, we also observed that there was no significant signal difference between the five proteins after exposing the PVDF membrane to the chemiluminescent reagent ( Figure 2 B) The results show that CA preincubation inactivates HRP but does not cause protein loss or antibody misrecognition on NC and PVDF blotting membranes. In contrast, CA preincubation not only stabilizes NC membrane-bound proteins but also improves the sensitivity of NC membrane detection (detecting small molecular weight proteins).

[0061] To investigate whether the inactivation of HRP by CA is suitable for sequential WB procedures, we treated NC blots with 20% CA under different conditions to test whether CA exhibits HRP inactivation effects in practical applications (40°C, 30 minutes). Figure 3 As shown in AB, the Atg7 signal was detected for the first time after the protein immunoblot membrane was pre-incubated with double-distilled water; then we treated the NC membrane with CA, and the Atg7 and p-JNK signals were detected for the second time. Figure 3As shown in AB. The results showed that pretreatment with double distilled water and then inactivation of HRP with CA had no significant effect on the protein signal intensity. However, when we pre-incubated the protein immunoblot membrane with CA and observed the Atg7 signal detected in the first detection, and then treated it with CA and performed a second detection, the imaging showed that the Atg7 band became thinner ( Figure 3 C), p-JNK band weakened ( Figure 3 D). The results indicate that two rounds of CA treatment hinder the detection of phosphorylated proteins and the same proteins as before. Furthermore, we compared the sensitivity of detection of p-JNK and JNK proteins from pre-incubated NC membranes. When pre-treated with double-distilled water, the NC membranes showed normal p-JNK signal intensity after exposure. After CA treatment, the blots were re-exposed to a clear background. Detection with a JNK antibody revealed normal JNK signal ( Figure 3 E). This is related to Figure 3 The results shown in Figures AB are consistent with the previous results. Similarly, we found that the first p-JNK signal detection after CA pretreatment was normal, and the background after the second exposure was also clear. JNK protein could also be re-detected after the second CA treatment of NC membranes ( Figure 3 F) Our results suggest that the order of detection can influence the results. Therefore, based on our conclusions, phosphorylated proteins should be detected first, followed by non-phosphorylated proteins under two rounds of CA treatment. This approach achieves the advantages of horseradish peroxidase inactivation and improved detection sensitivity.

[0062] To further confirm the above results, we performed three rounds of CA inactivation on HRP ( Figure 4 The results showed that under the same treatment conditions, the results of the first detection of the p-p38 MAPK band were significantly different between double distilled water pretreatment and CA pretreatment ( Figure 4 A). Then, the same NC blot was repeatedly incubated with CA for the experiment. After the first HRP inactivation and the second detection, a clear Atg7 band appeared in the CA group compared with the double-distilled water incubation ( Figure 4 B). Subsequently, we successfully detected casapse 3 ( Figure 4 C) and Vinculin protein bands ( Figure 4 D) Surprisingly, we are still Figure 4 The previous residual bands p-p38 MAPK (marked by #) and casapse 3 ( Figure 4D, solid triangles indicate that even after three cycles of 20% CA treatment, p-p38 MAPK and caspase-3 antibodies were not dissociated from the NC membrane. Furthermore, we observed that the NC membrane surface became less smooth after CA treatment. These results suggest that the inhibitory effect of 20% CA is reversible with increasing wash cycles. Therefore, more than three cycles of 20% CA treatment are not recommended.

[0063] 4.2 Summary

[0064] In this study, we found that 10% to 20% CA inactivated HRP and allowed for continued western blotting. To date, there has been no clear evidence that CA treatment leads to protein loss, membrane damage, or loss of antibody activity.

[0065] like Figure 4 As shown in Figures CD, we noticed several faint bands in the blots treated with repeated CA. In addition to the above inferences, we believe that the anti-rabbit secondary antibody may cross-react with the previous antibody, resulting in the reappearance of the previous bands. Therefore, to maintain a clear and bright background, we need to use a primary antibody from a different host species or an antibody from the same species but a different isotype.

[0066] Regarding the mechanism by which CA inhibits HRP, we hypothesize the following: 1) Hydrophobic and electrostatic interactions may promote protein binding to nitrocellulose membranes. Although repeated washing with milk and Tween 20 can be used to remove proteins, current studies have shown that the acidic buffers described in this application (10% CA [pH=1.63], 20% CA [pH=1.54]) not only inactivate HRP but also stabilize the interaction between proteins and the blot membrane ( Figure 1-4 ) and almost eliminated the adverse stripping effects of milk and Tween20. Unexpectedly, we observed increased binding of caspase 3 isoform (17 kDa) and phosphor Akt to NC blot membranes and showed enhanced assay signals ( Figure 2 These results suggest that 1) CA buffer at pH 1–2 stabilizes antigens and antibodies without causing loss of biological activity on the blot membrane. 2) Acidic buffers induce conformational changes and denaturation of HRP at pH 3–4, and that 10%–20% CA buffer (pH 1.5–1.63) inactivates HRP through the aforementioned mechanism. 3) HRP inactivation during CA incubation may be attributed to changes in the stability of its secondary and tertiary structures, similar to the effect of heating on enzyme activity.

[0067] Results showed that 20% CA can repeatedly inactivate HRP, allowing the NC membrane to be reused four times. Furthermore, this method offers unique advantages over other detection methods, such as increased sensitivity for small molecular weight and phosphorylated proteins. Therefore, the inventors believe that this new method, which allows for the reuse of blot membranes, will be popular in chemiluminescent protein immunoblotting.

[0068] 5. Citations

[0069] [1]Rebay, I., Fehon, RG, Cold Spring Harbor protocols 2011, 2011,pdb.prot4998.

[0070] [2]Wang, JL, Li, MQ, Zhang, JJ, Xu, CJ, Biotechnic &histochemistry: official publication of the Biological Stain Commission2021, 1-11.

[0071] [3] Wang, JL, Chen, WG, Zhang, JJ, Xu, CJ, Journal of molecular histology 2021, 52, 521-537.

[0072] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for using a protein immunoblotting membrane regeneration solution, characterized in that: The following steps are involved: Step (1): using a protein immunoblotting membrane, horseradish peroxidase, and chemiluminescence reagent to perform protein immunoblotting detection; Step (2): Washing off the chemiluminescent reagent on the protein immunoblotting membrane used in step (1); Step (3): adding the protein immunoblot membrane from which the chemiluminescent reagent was washed in step (2) to the protein immunoblot membrane regeneration solution for shaking and incubation; Step (4): taking out the membrane in step (2), washing it with double-distilled water and / or TBST solution to obtain a membrane containing inactivated horseradish peroxidase; The protein immunoblotting membrane regeneration solution formula includes crotonic acid, and the crotonic acid concentration is 10%-20% by mass.

2. The method for using the protein immunoblotting membrane regeneration solution according to claim 1, wherein: Also includes: Step (5): The membrane containing inactivated horseradish peroxidase is obtained by step (4), and protein immunoblotting is performed using horseradish peroxidase and chemiluminescence reagent.

3. The method for using the protein immunoblotting membrane regeneration solution according to claim 2, wherein: The protein detected in step (5) is not the same protein as the protein detected in step (1).

4. The method for using the protein immunoblotting membrane regeneration solution according to claim 1, wherein: Steps (1) to (4) are repeated at least twice.

5. The method for using the protein immunoblotting membrane regeneration solution according to claim 4, wherein: The proteins detected later are not the same as those detected previously.

6. The method for using the protein immunoblotting membrane regeneration solution according to claim 1, wherein: The solid phase support of the protein immunoblotting membrane is a polyvinylidene fluoride membrane or a nitrocellulose membrane.

7. The method for using the protein immunoblotting membrane regeneration solution according to claim 6, wherein: The solid phase support of the protein immunoblotting membrane is a nitrocellulose membrane.

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