A protein immunoblotting membrane removal solution and method thereof

Abstract

The present application belongs to the technical field of antigen-antibody detection, and particularly relates to a protein immunoblotting membrane removing liquid and a method thereof. The protein immunoblotting membrane removing liquid comprises trypsin. It is unexpectedly found that the trypsin can completely and efficiently remove the primary antibody-secondary antibody complex from the immunoblotting membrane, so that the original blotting can be removed, and the new antigen on the blotting membrane can be detected again, the result is reliable, and therefore the purpose of good reuse of the blotting membrane can be achieved. Through the method, multiple gel electrophoresis for detection of different targets is not needed, and therefore the waste of rare or expensive samples is avoided.

Description

Technical Field

[0001] This invention belongs to the field of antigen and antibody detection technology, specifically relating to a protein immunoblotting membrane removal solution and its method. Background Technology

[0002] Western blotting (WB) is a hybridization technique that combines high-resolution gel electrophoresis with immunochemical analysis. In simple terms, it involves electrotransferring proteins onto a solid phase using an electrophoresis gel, followed by antibody detection of protein expression. The experimental principle is as follows: protein samples are electrophoresed through a PAGE gel at a constant voltage or current. Due to the different charges carried by the molecular weights of the proteins, they are separated at different electrophoretic speeds. The solid phase is then electrotransferred onto a blot membrane, where proteins are adsorbed non-covalently. These proteins then bind to primary and secondary antibodies, and the resulting substrate is analyzed by chemiluminescence to detect the separated protein components or their expression levels.

[0003] In addition, to save time and money, the reuse of blot membranes has become an important issue of concern for researchers. Reuse refers to the ability to elute with stripping buffer after development and fixing, then block again to detect new antigens, continue incubation with primary and secondary antibodies, and then perform chemiluminescence development.

[0004] Currently, existing technologies report various stripping buffers for the reuse of blot membranes. Among them, methods for inactivating HRP activity include H2O2, sodium azide, 3,3-diaminobenzidine or Vector SG and acetic acid, but these methods have their own limitations. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a protein immunoblotting membrane removal solution and method thereof.

[0006] The technical solution adopted in this invention is as follows: a protein immunoblotting membrane removal solution, which includes trypsin.

[0007] It is prepared by dissolving 0.1%-0.25% trypsin in ddH2O at a pH of 7.0-7.4.

[0008] A method for removing protein immunoblotting membrane blots, comprising the following steps:

[0009] (1) Rinse the protein immunoblot membrane with ddH2O;

[0010] (2) Transfer the protein immunoblot membrane after rinsing in step (1) to the protein immunoblot membrane removal solution as described above, and treat it at 40°C for 30 min.

[0011] (3) Rinse the protein immunoblot membrane after step (2) with ddH2O.

[0012] The protein immunoblot membrane is repeatedly peeled off no more than twice using the protein immunoblot membrane removal solution described above.

[0013] The beneficial effects of this invention are as follows: This invention unexpectedly discovered that trypsin can thoroughly and efficiently remove the primary and secondary antibody complex from the immunoblot membrane, allowing the original blot to be cleared and enabling the re-detection of new antigens on the blot membrane with reliable results. Therefore, it achieves the goal of reusing the blot membrane effectively. This method eliminates the need for multiple gel electrophoresis sessions for detecting different targets, thus avoiding the waste of scarce or expensive samples. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0015] Figure 1 The results of the dot pattern method are shown in A, B, C, and D, which are comparisons before and after removal under different removal conditions using different removal solutions.

[0016] Figure 2 To compare the results of two treatment methods for the NC membrane after electrotransfer of proteins; one is the conventional method followed by antigen detection. Figure 2 (A; B) Another method is to treat the NC membrane with 0.1% trypsin for 30 minutes before proceeding with subsequent antigen detection. Figure 2 .C; D);

[0017] Figure 3 The effects of trypsin concentration (0.1%, 0.05%) and blot treatment time (10 min) on the stripping of the blot membrane were investigated. A and B showed the NC membrane after development treated with freshly prepared trypsin. C and D showed the NC membrane treated with trypsin that had already been stripped of the blot membrane.

[0018] Figure 4The effects of trypsin concentration (0.1%, 0.05%) and blot treatment time (30 min) on the stripping of the blot membrane are shown. A and B are NC membranes after Atg7 and caspase3 antibody development. C and D are NC membranes treated with trypsin and stripped with trypsin removal solution for 30 min, and then exposed again. E and F are the results of the second detection of the NC membranes treated with trypsin.

[0019] Figure 5 Experiments involving repeated peeling of the membrane with trypsin removal solution; A1; B1 are Atg7 NC membranes after development; A2; B2 are Atg7 NC membranes after one peeling treatment in the 30 min 0.1% group and the 30 min 0.25% group; A3; B3 are Atg7 NC membranes after one peeling followed by incubation with new antibody; A4; B4 are Atg7 NC membranes after two peeling treatments in the 30 min 0.1% group and the 30 min 0.25% group; A5; B5 are Atg7 NC membranes after two peelings followed by incubation with new antibody. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] 1. Experimental Materials and Methods

[0022] (1) Experimental materials:

[0023] Trypsin (Solepro, China); BeyoECL Star chemiluminescent substrate;

[0024] NC membrane with a pore size of 0.45 μm (Millipore, Massachusetts, United States)

[0025] Bradford Protein Assay Kit;

[0026] RIPA buffer (Beyotime Biotechnology, Shanghai, China);

[0027] Three-color protein-loaded markers (TOROIVD, Shanghai, China);

[0028] 4-week-old, 23-gram adult male C57BL / 6J mice, Gem Pharma tech Co., Ltd (License No.: SCXK-(SU)-2018-0008; Nanjing, China);

[0029] This study was ethically approved by the Medical Ethics Committee of Wenzhou Medical University. All experimental procedures were approved by the Animal Care and Use Committee of Wenzhou Medical University (No.: 2021-0020). Mice were kept at 24-25 degrees Celsius, with a 12 / 12-hour day / night cycle, and were provided with water and food.

[0030] Table 1. Materials and Sources

[0031]

[0032] (2) Experimental method:

[0033] Preparation of trypsin removal solution: 0.1%-0.25% trypsin, dissolved in ddH2O, pH 7.0-7.4. The prepared trypsin removal solution can be stored at -20°C for several months.

[0034] Instructions for using trypsin removal solution: Rinse the NC membrane with protein blots once with ddH2O for 5 minutes. Then transfer the NC membrane to a 50ml centrifuge tube containing removal solution of different concentrations. Place the 50ml centrifuge tube in a 40℃ incubator for 10 or 30 minutes. After that, remove the NC membrane. Rinse the NC membrane again for 5 minutes. Then proceed with the standard Western blotting procedure.

[0035] 2. Abbreviation

[0036] In this article, the abbreviations are as follows: room temperature, RT; horseradish peroxidase, HRP; nitrocellulose membrane, NC membrane.

[0037] 3. Implementation Methods

[0038] 3.1 Dot hybridization test

[0039] Diluted HRP-conjugated goat anti-rabbit IgG was prepared at a concentration of 80 ng / μL in 0.1% Tween 20 (TBST) buffered saline containing 5% skim milk. We then spotted 1 μL of the diluted antibody-HRP onto an NC membrane, air-dried it at room temperature for 30 minutes, and then eluted it with trypsin buffer at 40°C for 10 minutes and 30 minutes, respectively. Immediately after treatment, the membrane was washed in double-distilled water for 5 minutes and then immersed in double-distilled water until just before detection, at which point the enzyme substrate was added for analysis (protein side up).

[0040] like Figure 1 As shown, using the spot imprinting method, it was found that after HRP chemical development ( Figure 1A1; B1; C1; D1) were treated again with the removal solution at different time points (10 min; 30 min) and different concentrations (0.1%; 0.25%), followed by rinsing with double-distilled water every 5 min, and then exposed again. It was found that development was still possible. Figure 1 (A2; B2; C2; D2). These experimental results indicate that the membrane stripping mechanism of the trypsin removal solution is not related to the inactivation of HRP. We speculate that it likely works by directly removing the primary and secondary antibody complex on the NC membrane.

[0041] 3.2 WB Analysis

[0042] WB was performed using existing methods ([1] Wang, JL, Li, MQ, Zhang, JJ, Xu, CJ, A sensitive and reversible staining of proteins on blot membranes; Biotechnic & histochemistry: official publication of the Biological Stain Commission 2021, 1-11. [2] Wang, JL, Chen, WG, Zhang, JJ, Xu, CJ, Journal of molecular histology 2021, 52, 521-537.). Mouse livers were removed, cut into small pieces, and 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, leucine protein, pH=7.4; Beyotime Biotechnology [P0013B]). The fragments were then dispersed by sonication (VOSHIN-650W; Voshin, Wuxi, China) until the lysates became clear. The liver lysates were centrifuged at 13,000 g at 4°C for 30 minutes, and the supernatant was collected. Total protein concentration was determined using a protein assay kit (P0006C; Beyotime Biotechnology). Proteins (15 μg) were separated by polyacrylamide gel electrophoresis (Tris-glycine SDS-polyacrylamide) and transferred to an NC membrane. The membranes were placed in 5% skim milk dissolved in TBST at room temperature for 1 hour, washed with TBST for 1 hour, and incubated overnight at 4°C with primary antibodies (see Table 1). NC or PVDF membranes were then washed with TBST and incubated in TBST for 1 hour with HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG secondary antibodies (see Table 1), and the protein signals were visualized using a BeyoECL Star-enhanced chemiluminescence reagent. Finally, images at a quality of at least 600 dpi were obtained using an Amersham Imaging System 680 (GE Healthcare Biosciences, Uppsala, Sweden). Before re-detection, the membranes were washed with double-distilled water for 5 minutes to remove the chemiluminescent substrate and treated with elution buffers with different incubation times. Finally, all procedures were performed under identical conditions for parallel comparison.

[0043] The NC membrane after electrotransfer of proteins was processed using two methods. One method involved following a conventional approach with subsequent antigen detection. Figure 2 (A; B) Another method is to treat the NC membrane with 0.1% trypsin for 30 minutes before proceeding with subsequent antigen detection. Figure 2 (C; D). The results showed that vinculin and p53 could still be detected using conventional methods; however, neither vinculin nor p53 could be detected in the trypsin-treated group. Our experiments indicate that the trypsin removal solution should not be co-incubated with the protein-loaded NC blot membrane beforehand, otherwise, the protein will be completely lost.

[0044] To further explore the effects of the number of times trypsin was used, the concentration of trypsin, and the processing time of the blot membrane on the stripping of the blot membrane, we conducted the following experiment. The protein detection antibody used was vinculin (…). Figure 3 (A1-D2). For Figure 3 A1-B2, the trypsin was freshly prepared. After 10 minutes of trypsin treatment with the developed NC membrane, we found that trypsin of different concentrations failed to successfully peel off the NC membrane. Figure 3 (A2;B2), after exposure, the previous bands are still visible. However, when the NC membrane is treated with trypsin that has already stripped the imprint membrane (…),… Figure 3 (C1-D1), we found that even after treating the NC membrane with different concentrations of trypsin for 10 minutes, the exposed bands were still visible. Figure 3 (C2;D2). This indicates that a 10-minute treatment time with trypsin, whether freshly prepared or recovered after use, cannot successfully peel off the NC membrane.

[0045] Through the above experiments, we found that the 10-minute removal solution was insufficient for NC membrane peeling. Therefore, we further explored the effect of 30-minute incubation with the removal solution on NC membrane peeling. The NC membrane after development with Atg7 and caspase3 antibodies (…) Figure 4 After peeling with trypsin solution for 30 minutes, and then exposing again, the background of the imprinted membrane was found to be relatively clean, indicating that the peeling was successful. Figure 4 .CD). Then, after rinsing the NC membrane with ddH2O, the subsequent experiments continued according to the standard procedure. We found that in both the 30 min 0.25% and 30 min 0.1% removal buffer groups, the neoantigen signal could be detected again after successful NC membrane removal. Figure 4 (EF). This part of the experiment shows that a 30-minute incubation time is crucial for the successful peeling of the NC membrane by trypsin washing.

[0046] To determine the number of times the trypsin removal solution could be successfully repeated for membrane peeling, we continued the experiments with 0.1% for 30 min and 0.25% for 30 min. The results showed that both the 0.1% group and the 0.25% group could successfully peel the developed Atg7 NC membrane (…). Figure 5 .A1;B1) Successfully peeled off ( Figure 5 (A2;B2), and after further incubation of the new antibody, the vinculin band was found to be visible. Figure 5 A3; B3, the contamination is related to excessive antibody usage. We successfully peeled the membrane again. Figure 5 A4; B4), the background is clean. The peeled NC membrane was incubated with ERK1 / 2 antibody, and it was found that ERK1 / 2 could show (…). Figure 5 (A5; B5). However, the ERK1 / 2 band development in the 0.1% group at 30 min was slightly lighter than that in the 0.25% group at 30 min. Finally, this part of the experiment shows that our novel trypsin blot membrane removal solution can be repeatedly peeled up to twice.

[0047] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for removing protein immunoblotting membrane blots, characterized in that... Includes the following steps: (1) Rinse the protein immunoblot membrane with ddH2O; (2) Transfer the protein immunoblot membrane after rinsing in step (1) to the protein immunoblot membrane removal solution and treat it at 40°C for 30 min. (3) Rinse the protein immunoblot membrane after step (2) with ddH2O; The protein immunoblotting membrane removal solution includes trypsin; The protein immunoblotting membrane removal solution was prepared by dissolving 0.1%-0.25% trypsin in ddH2O, with a pH of 7.0-7.

4.

2. The method for removing protein immunoblotting membrane blots according to claim 1, characterized in that: The protein immunoblot membrane is repeatedly peeled off no more than twice using the protein immunoblot membrane removal solution.

Images