An immunoblot membrane eluent and its usage method

Abstract

The present invention provides an immunoblot membrane eluent and a method for using the same. The immunoblot membrane eluent comprises: 0.12% - 0.22% of tris(hydroxymethyl)aminomethane, 0.037% - 0.047% of EDTA, 0.005% - 0.01% of Tween 20. The above raw materials are dissolved in ddH2O, and the pH value is adjusted to 9.5 - 10.0. The present invention can not only remove the affinity-bound primary antibody and secondary antibody, but also enable the undetected target antigen to remain on the blot membrane, so as to achieve a good effect of reusing the blot membrane again. There is no need to perform multiple gel electrophoreses for detecting different targets. By this method, the NC membrane can be repeatedly eluted at least 5 times, and the detection signal of the new antigen remains unchanged, thus avoiding waste of rare or expensive samples.

Description

Technical Field

[0001] The present invention relates to the field of antibody detection, and particularly to an immunoblot membrane eluent and a method for using the same. Background Art

[0002] Since 1981 when Neill Burnette first called the protein immunoblotting method Western blotting (WB), this experimental technique has been widely used in research fields such as biochemistry and molecular biology. Western blotting, also known as immunoblotting, is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. This method is a new immunochemical technique developed on the basis of gel electrophoresis and solid-phase immunoassay techniques. The basic steps are as follows: according to the molecular weight of the protein, first use gel SDS-PAGE electrophoresis to separate the protein sample, and transfer it to a solid-phase carrier, such as nitrocellulose membrane (NC). The solid-phase carrier can adsorb proteins and keep the polypeptide types separated by electrophoresis and their biological activities unchanged. The transferred NC membrane is called a blot, which is treated with a protein solution (such as 5% BSA or skim milk powder solution) to block the hydrophobic binding sites on the NC membrane. Immediately afterwards, the primary antibody and the secondary antibody are incubated, and finally DAB or chemiluminescence method is used for development. This method has high resolution and high specificity and sensitivity of the antigen-antibody reaction in solid-phase immunoassay.

[0003] Performing gel electrophoresis and using parallel loading immunoblot analysis to detect new primary antibodies or antibody concentrations is very time-consuming and costly. Therefore, how to reuse the blot membrane has become a focus issue for researchers. Reuse means that after development and fixation are completed, it can be re-eluted (stripping buffer), then re-blocked to detect new antigens, and continue the incubation of the primary antibody (which can be different, provided that the target protein is on the blot membrane) and the secondary antibody, followed by development and fixation. Although a variety of methods for inactivating HRP activity for reusing the blot membrane have been reported, including H 2 O 2 , sodium azide, 3,3-diaminobenzidine or Vector SG and acetic acid, these methods have their own limitations. And many stripping buffers (translated into Chinese as: eluent; regeneration solution; removal solution) can cause the loss of proteins on the blot membrane, resulting in the disappearance of detection signals and misleading researchers. In addition, generally, the solid-phase carrier combined with proteins or antigens can only be used for one protein immunoblotting experiment. However, the binding of antigen and antibody is the binding of ionic bonds and hydrophobic bonds on the molecular surface, and the complex formed under certain conditions (such as strong acidity, H 2 O 2 etc.) is not firm and can be dissociated. Summary of the Invention

[0004] The object of the present invention is to overcome the disadvantages and deficiencies existing in the prior art, and to provide a protein immunoblot membrane regeneration solution and a method for using the same. The technical solution adopted by the present invention is as follows:

[0005] An immunoblot membrane eluent, the immunoblot membrane eluent comprising:

[0006] Tris (tris(hydroxymethyl)aminomethane) 0.12% - 0.22%;

[0007] EDTA (ethylenediaminetetraacetic acid) 0.037% - 0.047%;

[0008] Tween 20 0.005% - 0.01%;

[0009] Dissolve the above raw materials with ddH 2 O, and adjust the pH value to 9.5 - 10.0; This eluent can be stored at room temperature (RT) for several months.

[0010] There is also provided a method for using the above immunoblot membrane eluent:

[0011] After transferring the protein to the NC immunoblot membrane, then rinse the NC membrane once with ddH 2 O (5 min); Then transfer the NC membrane to a 50 ml centrifuge tube containing the eluent, and then place the 50 ml centrifuge tube in water for boiling (about 95 °C). After the water boils, start timing. After 10 - 25 min, the NC membrane can be taken out. Then rinse the NC membrane once again (5 min). Subsequently, continue the WB operation according to the conventional procedure.

[0012] The beneficial effects of the present invention are as follows: The present invention can not only remove the affinity - bound primary antibody and secondary antibody, but also enable the undetected target antigen to remain on the immunoblot membrane, thereby achieving a good effect of re - using the immunoblot membrane again. There is no need to perform multiple gel electrophoreses for detecting different targets. By this method, the NC membrane can be repeatedly eluted at least 5 times, and the detection signal of the new antigen remains unchanged, thus avoiding waste of rare or expensive samples. Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, obtaining other drawings based on these drawings still belongs to the scope of the present invention.

[0014] Figure 1For the NC membrane on which the electrotransferred protein has been completed in Western blotting, continue to incubate with vinculin primary antibody and secondary antibody, and finally perform chemiluminescence detection and imaging to obtain an image with a vinculin band ( Figure 1 .A1; Figure 1 .B1); then wash with the eluent for 10 min and 25 min respectively, then rinse with double-distilled water, and obtain the image after re-exposure ( Figure 1 .A2; Figure 1 .B2); after the eluted NC membrane is continuously processed according to the conventional Western blot operation method, obtain the image after re-exposure ( Figure 1 .A3; Figure 1 .B3), incubate the NC membrane treated with the eluent with vinculin primary antibody again, and obtain the image after chemical exposure ( Figure 1 .A4; Figure 1 .B4).

[0015] Figure 2 For the imaging of vinculin protein Western blot according to the same experimental method ( Figure 2 .A1; Figure 2 .B1); then rinse the NC membrane with double-distilled water for 5 min, and obtain the image after treatment with the eluent containing Tween 20 ( Figure 2 .A2; Figure 2 .B2); finally, perform the operation according to the conventional Western blot, and obtain the imaging of the Atg7 band after incubating with Atg7 primary antibody again ( Figure 2 .A3; Figure 2 .B3).

[0016] Figure 3 For the imaging obtained by normal Western blot operation ( Figure 3 .A1; Figure 3 .B1; Figure 3 .C1), the change of the band signals of different proteins after imaging after treatment with the eluent containing Tween 20 ( Figure 3 .A2; Figure 3 .B2; Figure 3 .C2).

[0017] Figure 4 For the imaging of the electrotransferred protein vinculin ( Figure 4 .A1), the image of the NC membrane of the electrotransferred protein vinculin after stripping the membrane 5 times respectively with the eluent containing Tween 20 ( Figure 4 .A2; Figure 4 .A4; Figure 4 .A6; Figure 4.A8; Figure 4 .A10), the developed images of the detection of new different types of antigens (Atg7, Caspase-3, UBA5, JNK, ERK1 / 2 respectively) after each stripping of the membrane (corresponding respectively to Figure 4 .A3; Figure 4 .A5; Figure 4 .A7; Figure 4 .A9; Figure 4 .A11).

[0018] Figure 5 For using the dot blot method, the developed images by the HRP chemical method ( Figure 5 .A1; Figure 5 .B1); Treat with the eluent containing Tween 20 for different times (10 min; 25 min) again, then rinse with double-distilled water, and then develop the images obtained by exposure ( Figure 5 .A2; Figure 5 .B2). Detailed implementation manner

[0019] 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.

[0020] The directional and positional terms mentioned in the present invention, such as "up", "down", "front", "back", "left", "right", "inside", "outside", "top", "bottom", "side", etc., are only the directions or positions with reference to the accompanying drawings. Therefore, the directional and positional terms used are for explaining and understanding the present invention, rather than limiting the protection scope of the present invention.

[0021] 1. Experimental materials and methods

[0022] Tris base (Solarbio, China); Tween-20 (Solarbio, China);

[0023] NC membrane with a pore size of 0.45 μm (Millipore, Massachusetts, United States)

[0024] BeyoECL Star chemiluminescent substrate;

[0025] Bradford protein detection kit;

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

[0027] 3-color protein loading marker (TOROIVD, Shanghai, China);

[0028] 23 - gram adult male C57BL / 6J mice at 4 weeks of age, Gem Pharma tech Co., Ltd (license production number: SCXK-(SU)-2018-0008; Nanjing, China);

[0029] This study was 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 (number: 2021-0020). The mice were maintained at 24 - 25 °C with a 12 / 12-hour light / dark cycle and provided with water and food.

[0030]

[0031] 2. Abbreviations

[0032] Room temperature, RT; Horseradish peroxidase, HRP; Nitrocellulose membrane, NC membrane.

[0033] 3. Implementation methods

[0034] 3.1 Dot blot assay

[0035] Diluted HRP-conjugated goat anti-rabbit IgG was prepared at a concentration of 80 ng / μL in buffered saline containing 5% non-fat milk (0.1% Tween 20 (TBST)). Then we spotted 1 μL of the diluted antibody-HRP on the NC membrane and air-dried it at room temperature for 30 minutes before treatment with the eluent for 10 min and 25 min respectively. After treatment, the membrane was immediately washed in double-distilled water for 5 minutes and soaked in double-distilled water until taken out before detection and added with enzyme substrate for detection (protein side up).

[0036] 3.2 WB analysis

[0037] WB was performed according to the existing method [1][2]. The mouse liver 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]), and then dispersed using ultrasound (VOSHIN-650W; Voshin, Wuxi, China) until the lysate became clear. The liver lysate was centrifuged at 13,000 g for 30 minutes at 4 °C and the supernatant was collected. The total protein concentration was determined using a protein assay kit (P0006C; Beyotime Biotechnology). Proteins (15 μg) were separated by polyacrylamide gel (Tris-glycine SDS-polyacrylamide) electrophoresis and transferred to an NC membrane. After the membrane was placed in 5% non-fat milk dissolved in TBST for 1 hour at room temperature, washed with TBST for 1 hour, and incubated overnight at 4 °C with the primary antibody (see Table 1). Then, the NC or PVDF membrane was washed with TBST and incubated in TBST with secondary antibodies of HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (see Table 1) for 1 hour, and the protein signal was visualized using BeyoECL Star enhanced chemiluminescence reagent. Finally, an image with a quality of at least 600 dpi was obtained using an Amersham imager 680 (GE Healthcare Biosciences, Uppsala, Sweden). Before re-detection, it was washed with double-distilled water for 5 minutes to remove the chemiluminescence substrate and treated with the eluent with different incubation times. Finally, for parallel comparison, all procedures were carried out under the same conditions.

[0038] 4. Example:

[0039] (1) Preparation of eluent: Dissolve 0.12% - 0.22% tris(hydroxymethyl)aminomethane, 0.037% - 0.047% EDTA, and 0.005% - 0.01% Tween 20 in ddH 2 O, and adjust the pH value to 9.5 - 10.0;

[0040] (2) Usage method: After transferring the protein to the NC blotting membrane, rinse the NC membrane with ddH 2 O once (5 min); then transfer the NC membrane to a 50 ml centrifuge tube containing the eluent, and then place the 50 ml centrifuge tube in water for boiling (about 95 °C). After the water boils, start timing. After 10 min or 25 min, the NC membrane can be taken out. Then rinse the NC membrane once again (5 min). Then the WB operation can be continued according to the conventional procedure.

[0041]

[0042] As shown in Table 2, after the NC membrane was eluted with an elution solution without Tween-20 added in Comparative Example 1, the same antigen was re-detected at different time points and compared; Figure 1 As shown, the NC membrane of the electrotransferred protein was completed in the western, and the vinculin primary antibody and secondary antibody were incubated, and finally the chemiluminescence detection was performed, and the vinculin band appeared ( Figure 1 .A1; Figure 1 .B1). Then the eluent treatment was carried out for 10min and 25min respectively. After the treatment, it was rinsed with double distilled water for 5min and then exposed again. It was found that there were still many black spots on the eluent 10min treatment group ( Figure 1 .A2), while the group treated with the eluent for 25 min had fewer black spots ( Figure 1 .B2). This indicates that treating the blotting membrane with the eluent for 25 minutes can make the membrane background cleaner. Then the eluted NC membrane was treated according to the conventional western blot operation method (blocking, TBST washing), and then exposed again. The results showed that the NC membrane background was very clean regardless of whether it was treated with the eluent for 10 minutes or 25 minutes ( Figure 1 .A3; Figure 1 .B3), we speculate that this may be related to the presence of Tween-20 (a non-ionic surfactant) in TBST. Finally, we incubated the NC membranes treated with the two groups of eluents with vinculin primary antibody again at 4°C. After chemical exposure and imaging the next day, the vinculin bands reappeared, and the signal intensity of the vinculin bands treated with the eluent seemed to be more significant than that of the vinculin bands not treated with the eluent ( Figure 1 .A4; Figure 1 .B4).

[0043] like Figure 2 As shown, after eluting the NC membrane with the eluent added with Tween-20 in Example 1, different antigens were re-detected and compared at different time points; according to the same experimental method in Comparative Example 1, after western blot development of vinculin protein ( Figure 2 .A1; Figure 2 .B1), the NC membrane was rinsed with double distilled water for 5 minutes, and then treated with an eluent containing Tween-20. It was found that the background of the 10-min and 25-min treatment groups was very clean ( Figure 2 .A2; Figure 2 .B2). Finally, the conventional western blot was performed and the Atg7 primary antibody was incubated again overnight. The next day, the Atg7 band was clearly developed on the NC membrane ( Figure 2 .A3;Figure 2 . B3). Therefore, it is inferred that adding Tween 20 to the eluent can significantly improve the use effect of the eluent.

[0044] After the eluent in Example 1 elutes the blotting membrane, detect the effect of the eluent on the protein content on the blotting membrane; as Figure 3 shown, in order to explore whether the eluent in Example 1 would cause the loss of proteins on the blotting membrane, we compared the changes in the band signals after different proteins were developed by parallel comparison between the normal western blot operation group ( Figure 3 . A1; Figure 3 . B1; Figure 3 . C1) and the eluent treatment group ( Figure 3 . A2; Figure 3 . B2; Figure 3 . C2). It was found that for the large molecular weight protein vinculin, the medium molecular weight protein p-JNK, and the small molecular weight protein caspase 3, the final band detection signals did not change. Therefore, it can be considered that the eluent containing Tween-20 will not cause the loss of proteins on the blotting membrane, and these proteins also include phosphorylated proteins.

[0045] In order to explore how many times the eluent containing Tween-20 can be used to strip the membrane repeatedly, after repeatedly eluting the NC membrane with the eluent containing Tween-20, detect the effect of the antigen signal again. As Figure 4 shown, we used the eluent to strip the NC membrane of the electrotransferred protein vinculin ( Figure 4 . A1) 5 times ( Figure 4 . A2; Figure 4 . A4; Figure 4 . A6; Figure 4 . A8; Figure 4 . A10), and after each stripping, new different types of antigen detections were carried out, namely Atg7, Caspase-3, UBA5, JNK, ERK1 / 2 ( Figure 4 . A3; Figure 4 . A5; Figure 4 . A7; Figure 4 . A9; Figure 4 . A11). The experimental results show that after the same NC membrane is repeatedly eluted with this eluent, new antigens can still be clearly detected, and the band background is clear and the antigen detection signal is normal. M represents the molecular weight.

[0046] Chemiluminescent immunoblotting detection with horseradish peroxidase substrates is the most common and sensitive method currently, but these chromogenic substrates do not precipitate and permanently bind to the surface of the blotting membrane.

[0047] The mechanism of the eluent eluting the blotting membrane in Example 1 of the present invention was explored using the dot blotting method, as Figure 5 shown. It was found that after developing by the HRP chemical method ( Figure 5 .A1; Figure 5 .B1), the eluent containing Tween-20 was used to treat for different times (10 min; 25 min) again, and then double-distilled water rinsing was continued once for 5 min, and then exposure was carried out again ( Figure 5 .A2; Figure 5 .B2). It was found that development was still possible. The experimental results of this part indicate that the film stripping mechanism of the eluent containing Tween-20 has nothing to do with the inactivation of HRP, and it is very likely to act by directly removing the primary antibody and secondary antibody on the NC membrane.

[0048] 5. Cited references

[0049] [1] Wang, J. L., Li, M. Q., Zhang, J. J., Xu, C. J., A sensitive and reversible staining of proteins on blot membranes; Biotechnic & histochemistry: official publication of the Biological Stain Commission 2021, 1-11.

[0050] [2] Wang, J. L., Chen, W. G., Zhang, J. J., Xu, C. J., Journal of molecular histology 2021, 52, 521-537.

[0051] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. An immunoblot membrane eluent, characterized in that, the immunoblot membrane eluent comprises: Tris 0.12% - 0.22%; EDTA 0.037% - 0.047%; Tween 20 0.005% - 0.01%; Dissolve the above raw materials with ddH 2 O and adjust the pH value to 9.5 - 10.

0.

2. A method for using an immunoblot membrane eluent, characterized in that: Step 1: Rinse the blotted membrane after transfer with ddH 2 2O; Step 2: Transfer the rinsed blot membrane to a container containing the immunoblot membrane eluent as described in claim 1, then heat the container in a water bath. After the water boils, start timing. Take the blot membrane after 10 - 25 minutes; Step 3: After rinsing the blot membrane obtained in Step 2, continue the WB operation according to the conventional procedure.

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