Immunoblotting protein detection method based on microglial cell AD pathology

Abstract

The invention discloses an immunoblotting protein detection method based on microglial cell AD pathology, which comprises the following steps: S1, obtaining a protein sample, dissolving the protein sample in a Laemmli sample buffer solution for electrophoresis, separating multiple layers of bands, and performing transfer printing; s2, transferring the multiple layers of strips onto a nitrocellulose membrane to form a blotting membrane, and immersing absorbent paper and a support pad into a transfer printing buffer solution for assembly to form a transfer printing interlayer group; s3, the transfer printing interlayer set is put into a transfer printing groove for transfer printing; s4, taking out the imprinted membrane, adding a primary antibody solution for recognition, and catalyzing and combining through a horse radish peroxidase labeled secondary antibody reagent; and S5, taking out the incubated blotting membrane, bagging the blotting membrane, contacting the blotting membrane with the film for exposure, scanning the film, and analyzing the molecular weight and the net optical density value. The detection sensitivity and specificity are improved, the key protein of the microglial cells under AD pathology can be accurately recognized and quantified, and reliable protein expression data are provided for AD research.

Description

Technical Field

[0001] The present invention relates to the technical field of microglial protein detection, and specifically relates to an immunoblot protein detection method based on the AD pathology of microglia. Background Art

[0002] In the field of neuroscience, the research on Alzheimer's disease (AD) has always been a hot topic and a difficult point. As the immune cells of the central nervous system, microglia play a crucial role in the pathological process of AD, and their abnormal functions are closely related to the occurrence and development of AD. Exploring the protein expression changes of microglia under AD pathology can accurately reveal the pathogenesis of AD, providing key theoretical support for the early diagnosis, effective treatment and drug development of the disease. However, existing detection technologies have many deficiencies when detecting proteins related to the AD pathology of microglia, and it is difficult to meet the urgent needs of scientific research and clinical practice for accurate and efficient detection. Therefore, it is extremely urgent to develop a new and highly targeted immunoblot protein detection method.

[0003] Traditional protein detection methods such as immunohistochemistry mainly bind labeled antibodies to antigens in tissue sections and observe the antigen distribution through a microscope. Its advantage is that it can intuitively present the localization information of proteins in tissue cells and has unique value in studying the spatial distribution of proteins. However, this method has obvious defects. Its quantitative analysis accuracy is poor, and it is difficult to accurately measure the subtle changes in protein expression levels; the operation process is complex and cumbersome, with extremely high requirements for sample preparation and the technical level of experimental personnel; the detection sensitivity is limited, and it is easy to miss the detection of low-expression proteins.

[0004] At present, some relatively advanced protein detection technologies, such as mass spectrometry-based proteomics technologies, can identify and quantify multiple proteins on a large scale at one time and are widely used in proteomics research. It can provide comprehensive protein information, which helps to discover new biomarkers and potential therapeutic targets. However, this technology has expensive equipment, high experimental costs, requires professional technical personnel for operation and maintenance; the data analysis is complex, with high requirements for bioinformatics knowledge and computing resources; and the pre-treatment requirements for samples are extremely strict, and small differences in the sample preparation process lead to large result deviations, limiting its application in some small laboratories and clinical rapid detections.

[0005] Therefore, the immunoblot protein detection technology based on the AD pathology of microglia disclosed in this application effectively makes up for the deficiencies of the above-mentioned traditional and existing technologies. Summary of the Invention

[0006] Based on the above problems, this application proposes an immunoblot protein detection method based on the AD pathology of microglia, specifically as follows:

[0007] S1. Obtain a protein sample, dissolve the protein sample in Laemmli sample buffer, perform electrophoresis through SDS polyacrylamide gel to separate multiple bands, and then perform transfer;

[0008] S2. Transfer the multiple bands on the SDS polyacrylamide gel to a nitrocellulose membrane, soak it in transfer buffer to form a blotting membrane, and immerse blotting paper and a support pad in the transfer buffer for assembly to form a transfer sandwich group;

[0009] S3. Place the transfer sandwich group in a transfer tank for transfer. After completion, remove the support pad and blotting paper in the transfer sandwich group, wash the blotting membrane with PBS and incubate it;

[0010] S4. During the incubation process, take out the blotting membrane, add a primary antibody solution for recognition, and catalyze and bind it with a horseradish peroxidase-labeled secondary antibody reagent;

[0011] S5. Take out the incubated blotting membrane, incubate it in a chemiluminescent solution, after completion, put it in a bag and expose it to a film, scan the film, and analyze the molecular weight and net optical density value.

[0012] Preferably, the obtaining of the protein sample in S1 includes:

[0013] S1.1. Collect a sample containing microglia, where the sample is derived from the brain tissue or cell culture of AD-induced animals;

[0014] S1.2. Treat the sample with a lysis solution, where the lysis solution contains a protease inhibitor;

[0015] S1.3. After incubation, separate the supernatant by high-speed centrifugation to obtain a protein sample containing the target protein.

[0016] Preferably, add the protein sample to the SDS polyacrylamide gel, and under the action of an electric field through an electrophoresis device, the protein sample starts to migrate and separate into multiple bands in the SDS polyacrylamide gel. After electrophoresis is completed, remove the flat plate, cut the SDS polyacrylamide gel, and perform transfer after cutting is completed.

[0017] Preferably, before cutting, it is necessary to mark the SDS polyacrylamide gel to determine the direction, by marking small dots or short lines at the upper left corner or an easily recognizable position of the gel to help identify the direction of the gel during the transfer process and prevent misalignment of protein bands caused by incorrect direction.

[0018] Preferably, the assembly sequence of the transfer sandwich group in S2 is specifically as follows: support pad, absorbent paper, gel, blotting membrane, absorbent paper, and support pad; the support pad is placed at the bottom, the absorbent paper is laid flat above the support pad to adsorb the transfer buffer, the electrophoresed and marked gel is placed on the absorbent paper, the blotting membrane is covered on the gel to complete the transfer and fixation of the protein bands, and absorbent paper is placed to ensure the circulation and uniform distribution of the buffer. The support pad is placed on the top layer to form the transfer sandwich group.

[0019] Preferably, when the transfer sandwich group in S3 is placed in the transfer tank, the blotting membrane is positioned close to the positive electrode, so that the proteins in the gel are transferred to the blotting membrane, and transferred in the transfer tank at 3 - 5 °C for 6 - 8 h.

[0020] Preferably, after the transfer is completed, disconnect the power supply, remove the support pad and absorbent paper in the transfer sandwich group, make a mark on the blotting membrane, keep it in the direction before transfer. After marking, wash the blotting membrane with pre-cooled PBS solution to remove the residual transfer buffer and impurities on the surface of the blotting membrane. Put the washed blotting membrane into the prepared 5% skim milk powder / PBS solution and slowly agitate and incubate it at room temperature for 1 - 2 hours using a magnetic stirrer or other equipment.

[0021] Preferably, during the incubation in S4, take out the blotting membrane and rinse it with PBS solution to remove the unbound blocking reagent, and add the primary antibody solution; the primary antibody solution is a specific antibody for the target protein, diluted according to the set concentration, which can recognize the target protein and will not produce excessive background signals. For a 15 cm × 15 cm blotting membrane, 10 - 12 ml of the primary antibody solution is used; after adding the primary antibody solution, incubate it at 15 - 25 °C for 1 - 1.5 h to form the antigen - antibody complex.

[0022] Preferably, after the incubation and rinsing of the primary antibody solution are completed, add the horseradish peroxidase - labeled secondary antibody reagent; the horseradish peroxidase - labeled secondary antibody reagent will specifically recognize and bind to the Fc segment of the primary antibody solution; take the horseradish peroxidase - labeled secondary antibody reagent and add it to the incubation container, completely immerse the blotting membrane in the secondary antibody reagent, and agitate and incubate it using a low - speed magnetic stirrer or oscillation equipment to bind to the primary antibody that has already bound to the target protein on the membrane, forming the antigen - primary antibody - secondary antibody complex.

[0023] Preferably, in S5, a chemiluminescent reagent is prepared, and the treated blotting membrane is placed in the chemiluminescent reagent and incubated for 1 - 2 min. After the incubation, the excess chemiluminescent reagent at the edges or corners of the blotting membrane is blotted off. The blotting membrane is then placed flat in a plastic bag, and the surface of the dry blotting membrane is closely contacted with the film to transfer the chemiluminescent signal to the film. The plastic bag containing the blotting membrane and the film is transferred to a darkroom and exposed for 1 - 2 min in an environment free from light interference. After the exposure is completed, the film is quickly taken out, and the film is scanned using a scanner or photographed using a camera. The obtained image is analyzed using a gel image processing device to measure the molecular weight and net optical density value of the target band.

[0024] Compared with the prior art, the technical solution of the present application has the following technical effects:

[0025] By optimizing the steps for obtaining the protein sample, that is, collecting samples from AD-induced animal brain tissues or cell cultures, treating with a lysis buffer containing protease inhibitors and centrifuging at high speed to separate the supernatant, the present invention solves the problems of low protein purity and easy degradation in the sample, and obtains a target protein sample with high purity and good integrity, laying a foundation for subsequent accurate detection and ensuring that the experimental results can truly reflect the expression of microglial AD pathology-related proteins.

[0026] By using the technical solution of making marks before cutting the SDS polyacrylamide gel to determine the direction, assembling the transfer sandwich group (support pad, blotting paper, gel, blotting membrane, blotting paper, and support pad) in a specific order, and transferring the blotting membrane close to the positive electrode at an appropriate temperature, the present invention solves the problems of easy misalignment and low transfer efficiency during the protein band transfer process, realizes the efficient and accurate transfer of protein bands from the gel to the blotting membrane, ensures the accuracy and reliability of immunoblotting detection, and improves the experimental success rate.

[0027] By strictly standardizing the use and incubation conditions of the primary antibody solution (selecting a specific antibody according to the target protein and diluting it at a set concentration) and the horseradish peroxidase-labeled secondary antibody reagent (specifically recognizing the Fc segment of the primary antibody) (appropriate amount of reagent corresponding to a specific membrane area, appropriate temperature, and time), the present invention solves the problems of strong non-specific binding of antibodies and low detection sensitivity in traditional detection methods, enhances the recognition specificity and detection sensitivity of the target protein, can effectively detect low-expression microglial AD pathology-related proteins, and improves the detection effect.

[0028] The present invention adopts a technical solution of precisely formulating chemiluminescent reagents, controlling the incubation time of the blotting membrane, properly treating the blotting membrane after incubation and standardizing exposure, scanning or photographing in a darkroom, and analyzing using a gel image processing system and statistical software, which solves the problems of inaccurate data and difficult quantitative comparison in result analysis, realizes the precise measurement and statistical analysis of the molecular weight and net optical density value of the target band, provides reliable quantitative data support for the study of microglial AD pathology, and helps to deeply study the change law of protein expression.

[0029] The above description is only an overview of the technical solution of the present application. In order to be able to understand the technical means of the present application more clearly, so as to be implemented in accordance with the content of the specification, and in order to make the above and other purposes, features and advantages of the present application more obvious and understandable, the following takes the preferred embodiments of the present application and combines with the drawings to describe in detail as follows.

[0030] According to the following detailed description of the specific embodiments of the present application in combination with the drawings, those skilled in the art will understand the above and other purposes, advantages and features of the present application more clearly. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to these drawings. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual ratio.

[0032] Figure 1 Flowchart of the immunoblot protein detection method based on microglial AD pathology of the present invention;

[0033] Figure 2 Flowchart of obtaining immunoblot protein samples based on microglial AD pathology of the present invention;

[0034] Figure 3 Basic diagram of immunoblot protein sample bands based on microglial AD pathology of the present invention;

[0035] Figure 4 Specific binding diagram of immunoblot protein samples based on microglial AD pathology of the present invention;

[0036] Figure 5 Scanning diagram of immunoblot protein samples based on microglial AD pathology of the present invention;

[0037] Figure 6, Comparative diagram of immunoblot protein samples of the experimental group and the control group based on the AD pathology of microglia. Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some but not all of the embodiments of this application. In the following description, specific details such as specific configurations and components are provided only to assist in a comprehensive understanding of the embodiments of this application. Therefore, those skilled in the art should clearly understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Additionally, descriptions of known functions and structures are omitted for clarity and conciseness.

[0039] It should be understood that the "one embodiment" or "this embodiment" mentioned throughout the specification means that a specific feature, structure, or characteristic related to the embodiment is included in at least one embodiment of this application. Therefore, the "one embodiment" or "this embodiment" that appears throughout the specification does not necessarily refer to the same embodiment. In addition, these specific features, structures, or characteristics can be combined in one or more embodiments in any suitable manner.

[0040] In addition, this application may repeat reference numerals and / or letters in different instances. This repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or arrangements discussed.

[0041] The term "and / or" in this document is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, B exists alone, and both A and B exist simultaneously. The term " / and" in this document describes another association relationship of associated objects, indicating that two relationships may exist. For example, A / and B may represent: A exists alone, and both A and B exist. Additionally, the character " / " in this document generally indicates that the associated objects before and after are in an "or" relationship.

[0042] The term "at least one" in this document is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, at least one of A and B may represent: A exists alone, both A and B exist simultaneously, and B exists alone.

[0043] It should also be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion.

[0044] Example 1

[0045] This example mainly describes a method for detecting immunoblot proteins based on the AD pathology of microglia, as Figure 1 shown, including the following steps:

[0046] S1. Obtain a protein sample, dissolve the protein sample in Laemmli sample buffer, perform electrophoresis through SDS polyacrylamide gel, separate multiple bands, and perform transfer;

[0047] S2. Transfer the multiple bands on the SDS polyacrylamide gel to a nitrocellulose membrane, immerse it in transfer buffer for soaking to form a blot membrane, and immerse blotting paper and a support pad in the transfer buffer for assembly to form a transfer sandwich group;

[0048] S3. Place the transfer sandwich group in a transfer tank for transfer. After completion, remove the support pad and blotting paper in the transfer sandwich group, wash the blot membrane with PBS and incubate it;

[0049] S4. During the incubation process, take out the blot membrane, add a primary antibody solution for recognition, and catalyze and bind it with a secondary antibody reagent labeled with horseradish peroxidase;

[0050] S5. Take out the blot membrane after incubation, place it in a chemiluminescent solution for incubation. After completion, put it in a bag and contact it with a film for exposure, scan the film, and analyze the molecular weight and net optical density value.

[0051] Furthermore, the acquisition of the protein sample in S1, as Figure 2 shown, includes:

[0052] S1.1. Collect a sample containing microglia, where the sample is derived from the brain tissue of AD-induced animals or cell cultures;

[0053] S1.2. Treat the sample with a lysis solution, where the lysis solution contains a protease inhibitor;

[0054] S1.3. After incubation, separate the supernatant by high-speed centrifugation to obtain a protein sample containing the target protein.

[0055] Further, add the protein sample to the SDS polyacrylamide gel, and under the action of an electric field through an electrophoresis apparatus, the protein sample starts to migrate and separate into multiple bands in the SDS polyacrylamide gel. After electrophoresis is completed, remove the plate, cut the SDS polyacrylamide gel, and perform transfer after cutting is completed.

[0056] Further, before cutting, it is necessary to mark the SDS polyacrylamide gel to determine the direction. By marking small dots or short lines in the upper left corner or an easily recognizable position of the gel, it helps to distinguish the direction of the gel during the transfer process and prevent misalignment of protein bands due to incorrect direction.

[0057] Further, the assembly order of the transfer sandwich group in S2 is specifically: support pad, blotting paper, gel, blotting membrane, blotting paper, and support pad; the support pad is placed at the bottom, spread the blotting paper on top of the support pad to adsorb the transfer buffer, place the electrophoresed and marked gel on the blotting paper, cover the blotting membrane on the gel to complete the transfer and fixation of the protein bands, and place blotting paper to ensure the circulation and uniform distribution of the buffer. Place the support pad on the top layer to form the transfer sandwich group.

[0058] Further, when the transfer sandwich group is placed in the transfer tank in S3, make the blotting membrane close to the positive electrode position, so that the proteins in the gel are transferred to the blotting membrane, and transfer in the transfer tank at 3 - 5 °C for 6 - 8 h.

[0059] Further, after transfer is completed, disconnect the power supply and remove the support pad and blotting paper in the transfer sandwich group, mark the blotting membrane, keep the direction before transfer, and after marking, wash the blotting membrane with pre-cooled PBS solution to remove the residual transfer buffer and impurities on the surface of the blotting membrane. Put the washed blotting membrane into the prepared 5% skim milk / PBS solution and slowly agitate and incubate at room temperature for 1 - 2 hours using a magnetic stirrer or other equipment.

[0060] Further, during the incubation in S4, take out the blotting membrane and rinse it with PBS solution to remove the unbound blocking reagent, and add the primary antibody solution; the primary antibody solution is a specific antibody for the target protein, diluted according to the set concentration, which can recognize the target protein and will not produce excessive background signals. For a 15 cm × 15 cm blotting membrane, take 10 - 12 ml of the primary antibody solution; after adding the primary antibody solution, place it at 15 - 25 °C for incubation, and the incubation time is 1 - 1.5 h to form an antigen - antibody complex.

[0061] Further, after completing the incubation and rinsing of the primary antibody solution, add the horseradish peroxidase-labeled secondary antibody reagent; the horseradish peroxidase-labeled secondary antibody reagent will specifically recognize and bind to the Fc segment of the primary antibody solution; take the horseradish peroxidase-labeled secondary antibody reagent and add it to the incubation container, completely immerse the blotting membrane in the secondary antibody reagent, and use a low-speed magnetic stirrer or oscillating device to stir and incubate it to bind to the primary antibody that has already bound to the target protein on the membrane, forming an antigen-primary antibody-secondary antibody complex.

[0062] Further, prepare the chemiluminescent reagent in S5, place the treated blotting membrane into the chemiluminescent reagent, incubate for 1 - 2 min. After the incubation ends, aspirate the excessive chemiluminescent reagent at the edges or corners of the blotting membrane, place the blotting membrane flatly into a plastic bag, make the surface of the dry blotting membrane closely contact with the film, so that the chemiluminescent signal is transferred to the film. Transfer the plastic bag containing the blotting membrane and the film to a darkroom, expose it for 1 - 2 min in an environment without light interference. After the exposure is completed, quickly take out the film, scan the film using a scanner or take a photo with a camera, and analyze the obtained image using a gel image processing device to measure the molecular weight and net optical density value of the target band.

[0063] In this embodiment, it is described in detail that in the sample source of the present application, microglia in the animal brain tissue or cell culture induced by AD are selected to ensure the specificity of the research object. High-purity protein samples are obtained through optimized lysis solution treatment and high-speed centrifugation to reduce impurity interference; in the electrophoresis and transfer steps, the conditions and operation steps are precisely controlled, such as marking the direction before gel cutting, reasonably assembling the transfer sandwich group and controlling the transfer temperature and time, etc., to ensure the accuracy and efficiency of protein band transfer. During the antibody incubation process, the use and incubation conditions of the primary antibody and the secondary antibody are strictly standardized, significantly improving the detection sensitivity and specificity, and can accurately identify and quantify the key proteins of microglia under AD pathology, providing reliable protein expression data for AD research, and strongly promoting the research process of AD pathological mechanisms and related drug development.

[0064] Based on Example 1, in this embodiment, during the incubation process of S4, take out the blotting membrane, add the primary antibody solution for recognition, and catalyze and bind it with the horseradish peroxidase-labeled secondary antibody reagent. Specifically:

[0065] Take out the blotting membrane and rinse it with PBS solution. Perform the rinsing operation twice, with each rinse lasting for 5 minutes, to ensure that unbound blocking reagent is thoroughly washed away, providing a pure membrane surface environment for subsequent antibody binding; add the primary antibody solution. The preparation of the primary antibody solution requires precise selection of the corresponding specific antibody according to the target protein being studied and dilution at an appropriate concentration to ensure that it can effectively recognize the target protein in subsequent reactions without generating excessive background signals; for a 15 cm × 15 cm blotting membrane, 10 ml of the primary antibody solution is used. This dosage has been optimized through a large number of experiments and can ensure uniform distribution and full coverage of the antibody on the membrane surface. After adding the primary antibody solution, place the blotting membrane and the antibody at room temperature and incubate them using a gentle agitation device for 1 hour. During this period, antibody molecules will continuously move in the solution and specifically bind to the target protein on the membrane, forming a stable antigen-antibody complex. The agitation operation helps to accelerate this binding process, making it more sufficient and uniform, thus providing a reliable basis for subsequent detection steps. After the incubation is completed, rinse the membrane with PBS solution again. This time, it needs to be rinsed 4 times, with each liquid change and continuous rinsing for 5 minutes, to remove unbound primary antibody and avoid its interference with subsequent experimental results.

[0066] After completing the primary antibody incubation and rinsing, it enters the crucial step of adding the secondary antibody reagent labeled with horseradish peroxidase (HRP). Horseradish peroxidase is a commonly used enzyme label that has high catalytic activity and can catalyze specific substrates to undergo chemical reactions, generating detectable signals; in immunoblotting experiments, the HRP-labeled secondary antibody reagent will specifically recognize and bind to the Fc segment of the primary antibody. The selection of the secondary antibody is crucial and it is necessary to ensure its compatibility with the source species of the primary antibody. For example, if the primary antibody is a rabbit-derived antibody, then an anti-rabbit IgG secondary antibody should be selected to ensure effective binding between the secondary antibody and the primary antibody; according to the size of the blotting membrane and the requirements of the experimental system, accurately measure an appropriate amount of the HRP-labeled secondary antibody reagent and add it to the incubation container. Immerse the blotting membrane completely in the secondary antibody reagent, place it at room temperature, and agitate and incubate it using a low-speed magnetic stirrer or a gentle oscillation device for 1 hour. During this period, the secondary antibody molecules will continuously move in the solution and tightly bind to the primary antibody that has already bound to the target protein on the membrane, thus constructing a stable antigen-primary antibody-secondary antibody complex.

[0067] After the incubation is completed, in order to remove the unbound secondary antibody reagent and prevent its interference with subsequent detection results, the membrane needs to be rinsed with PBS. Strictly follow the standard operation and rinse it 4 times, with each liquid change followed by continuous rinsing for 5 minutes to ensure that impurities and excess secondary antibody on the membrane surface are thoroughly removed, making full preparations for the subsequent chemiluminescence detection step and ensuring the accuracy and reliability of the final experimental results.

[0068] In this embodiment, the blotting membrane is taken out and a primary antibody solution is added. The primary antibody, by virtue of its specificity, accurately identifies and binds to the target protein on the membrane, laying a foundation for subsequent detection. Then, a secondary antibody reagent labeled with horseradish peroxidase is added. The secondary antibody can specifically bind to the Fc segment of the primary antibody to form a stable antigen-primary antibody-secondary antibody complex. Horseradish peroxidase has efficient catalytic activity. When a chemiluminescent substrate is subsequently added, it can catalyze a chemical reaction of the substrate to generate a detectable signal, thereby greatly improving the detection sensitivity, allowing extremely small amounts of target proteins to be accurately detected, and ensuring the accuracy and reliability of the results of the immunoblotting experiment.

[0069] Example 2

[0070] This paper describes in detail the immunoblotting protein detection method based on microglial AD pathology, aiming to explore the expression changes of microglia-related proteins in the brain of Alzheimer's disease (AD) model mice, specifically:

[0071] C57BL / 6 mice treated with specific AD inducers were selected as research subjects. Brain tissues were collected at 3, 6, and 9 months after induction. The brain regions containing microglia were quickly separated after dissection, and the tissue samples were cut into small pieces and placed in pre-cooled lysis buffer; the lysis buffer contained 1% Triton X-100, 150mM NaCl, 50mM Tris-HCl (pH7.4) and protease inhibitor cocktail (Roche, 1:100) to ensure that the protein was not degraded during the 30-minute incubation on ice. Subsequently, the supernatant was collected by high-speed centrifugation at 12000rpm at 4°C for 20 minutes, and the protein concentration was determined using a BCA protein quantification kit (Thermo Scientific) to ensure that the protein amount of each sample was consistent and met the requirements of subsequent experiments.

[0072] According to the molecular weight of the protein, select an SDS polyacrylamide gel of appropriate concentration for electrophoresis, such as 10% separation gel and 5% stacking gel. Mix 50 μg of protein sample with 5× Laemmli sample buffer, heat at 95°C for 5 minutes to denature the protein, load the sample into the sample well, and perform stacking gel electrophoresis at 80V. After the sample enters the separation gel, increase the voltage to 120V and perform electrophoresis for about 90 minutes until the bromophenol blue indicator migrates to the bottom of the gel to ensure that the protein bands are fully separated, providing a clear band basis for subsequent transfer, such as Figure 3 shown.

[0073] Transfer was carried out using a 0.45 μm nitrocellulose membrane and the wet transfer method. The transfer buffer was 25 mM Tris, 192 mM glycine, 20% methanol (pH 8.3). Assemble the gel, nitrocellulose membrane and filter paper in a sandwich structure, ensure no air bubbles, place it in the transfer tank, make the nitrocellulose membrane close to the positive electrode, and transfer at 100 V for 90 minutes at 4°C. After the transfer is completed, rinse the nitrocellulose membrane 3 times with pre-cooled PBS for 5 minutes each time to remove residual buffer and impurities;

[0074] Put the nitrocellulose membrane into a 5% skim milk / PBS solution and incubate it with shaking at room temperature for 2 hours to block non-specific binding sites. Subsequently, rinse it 3 times with PBS for 5 minutes each time, add the primary antibody solution against the microglial marker protein Iba-1 (1:1000, Abcam). For a 15 cm × 15 cm blot membrane, use 10 ml of the primary antibody solution and incubate it with shaking at room temperature for 1.5 hours. After the incubation, rinse it 4 times with PBS for 5 minutes each time, then add the horseradish peroxidase-labeled secondary antibody reagent (1:5000, Jackson ImmunoResearch), and also incubate it with shaking at room temperature for 1 hour. Finally, rinse it 4 times with PBS for 5 minutes each time to ensure a clean background and only retain specific binding signals, as Figure 4 shown.

[0075] Prepare fresh chemiluminescence reagent (ECL, Thermo Scientific), put the nitrocellulose membrane into it and incubate for 1.5 minutes, suck off the excess reagent, put it into a plastic bag and closely contact it with the film, expose it in the darkroom for 1.5 minutes, and scan the exposed film with a scanner, as Figure 5 shown, and use ImageJ software to analyze the bands.

[0076] Obtain the analysis results, as Figure 6 shown. At 3 months after AD induction, the expression level of Iba-1 protein increased by 23.65% compared with the control group (n = 5, p < 0.05), at 6 months it increased by 42.45% (n = 5, p < 0.01), and at 9 months it increased by 67.86% (n = 5, p < 0.001), indicating that microglia are gradually activated and the number increases during the AD process, which is consistent with the expected pathological changes, highlighting that the method of this application can accurately detect protein expression changes and provide strong technical support for AD research.

[0077] The immunoblot protein detection method of this example can accurately detect the changes in the expression of Iba-1 protein at different time points in AD research. The data show that its expression level is significantly increased compared with the control group at 3, 6, and 9 months after AD induction, which strongly reveals the activation and proliferation trend of microglia during the AD process and provides key data support for AD pathological research.

[0078] The above are only the preferred embodiments of the present invention, and thus do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any changes, modifications, substitutions, integrations, and parameter alterations made to these embodiments within the spirit and principle of the present invention, by means of conventional substitutions or capable of achieving the same functions without departing from the principle and spirit of the present invention, shall fall within the protection scope of the present invention.

Claims

1. A method for detecting microglial AD pathology based on immunoblotting protein, characterized in that: include: S1. Obtain a protein sample, dissolve the protein sample in Laemmli sample buffer, perform electrophoresis on SDS polyacrylamide gel, separate multiple bands, and transfer; S2, transferring the multilayer bands on the SDS polyacrylamide gel to a nitrocellulose membrane, and soaking it in a transfer buffer to form a blot membrane, and immersing the absorbent paper and the support pad in the transfer buffer to assemble them to form a transfer sandwich group; S3, placing the transfer sandwich group into the transfer tank for transfer, removing the support pad and absorbent paper in the transfer sandwich group after the transfer is completed, washing the blotting membrane with PBS and incubating; S4, during the incubation process, the blot membrane is taken out and the primary antibody solution is added for identification, and the secondary antibody reagent labeled with horseradish peroxidase is used for catalysis and binding; S5. Take out the blot membrane after the incubation is completed, place it in a chemiluminescent solution for incubation, put it in a bag and contact it with a film for exposure, scan the film, and analyze the molecular weight and net optical density value.

2. The immunoblotting protein detection method based on microglial AD pathology according to claim 1, characterized in that: The acquisition of protein samples in S1 includes: S1.

1. Collect samples containing microglia, wherein the samples are derived from AD-induced animal brain tissue or cell culture; S1.2, treating the sample with a lysis buffer, wherein the lysis buffer contains a protease inhibitor; S1.

3. After incubation, separate the supernatant by high-speed centrifugation to obtain a protein sample containing the target protein.

3. The immunoblotting protein detection method based on microglial AD pathology according to claim 2, characterized in that: The protein sample is added to the SDS polyacrylamide gel, and the protein sample begins to migrate and separate into multiple layers of bands in the SDS polyacrylamide gel under the action of an electric field through an electrophoresis device. After the electrophoresis is completed, the plate is removed, the SDS polyacrylamide gel is cut, and after the cutting is completed, transfer printing is performed.

4. The immunoblotting protein detection method based on microglial AD pathology according to claim 3 is characterized in that Before cutting, it is necessary to mark the SDS polyacrylamide gel to determine the direction. By marking a small dot or short line in the upper left corner of the gel or an easily identifiable position, it helps to identify the direction of the gel during the transfer process to prevent the protein band from being misplaced due to incorrect direction.

5. The immunoblotting protein detection method based on microglial AD pathology according to claim 1, characterized in that The assembly order of the transfer sandwich group in S2 is specifically: support pad, absorbent paper, gel, blotting membrane, absorbent paper and support pad; the support pad is placed at the bottom, the absorbent paper is spread flat on the support pad to absorb the transfer buffer, the electrophoresed and marked gel is placed on the absorbent paper, the blotting membrane is covered on the gel, so that the protein bands are transferred and fixed, and the absorbent paper is placed to ensure the circulation and uniform distribution of the buffer solution, and the support pad is placed on the top layer to form a transfer sandwich group.

6. The immunoblotting protein detection method based on microglial AD pathology according to claim 1, characterized in that When the transfer sandwich group in S3 is placed in the transfer tank, the blotting membrane is placed close to the positive electrode so that the protein in the gel is transferred to the blotting membrane and transferred in the transfer tank at 3-5°C for 6-8h.

7. The immunoblotting protein detection method based on microglial AD pathology according to claim 6 is characterized in that After the transfer is completed, disconnect the power supply, remove the support pad and absorbent paper in the transfer sandwich group, mark the blotting membrane, and keep it in the direction before transfer. After marking, wash the blotting membrane with pre-cooled PBS solution to remove the transfer buffer and impurities remaining on the surface of the blotting membrane. Place the washed blotting membrane in the prepared 5% skimmed milk powder / PBS solution, and incubate it for 1-2 hours at room temperature with a magnetic stirrer or other equipment.

8. The immunoblotting protein detection method based on microglial AD pathology according to claim 1, characterized in that During the S4 incubation process, the blotting membrane is taken out and rinsed with PBS solution to remove unbound blocking reagent, and a primary antibody solution is added; the primary antibody solution is a specific antibody for the target protein, which is diluted according to the set concentration to recognize the target protein without generating excessive background signals. For a 15cm×15cm blotting membrane, 10-12ml of the primary antibody solution is used; after adding the primary antibody solution, it is placed at 15-25°C for incubation for 1-1.5h to form an antigen-antibody complex.

9. The immunoblotting protein detection method based on microglial AD pathology according to claim 8, characterized in that After the incubation and rinsing of the primary antibody solution are completed, a secondary antibody reagent labeled with horseradish peroxidase is added; the secondary antibody reagent labeled with horseradish peroxidase will specifically recognize and bind to the Fc segment of the primary antibody solution; the secondary antibody reagent labeled with horseradish peroxidase is added to the incubation container, the blot membrane is completely immersed in the secondary antibody reagent, and a low-speed magnetic stirrer or oscillation device is used for stirring and incubation, so that it binds to the primary antibody that has been bound to the target protein on the membrane to form an antigen-primary antibody-secondary antibody complex.

10. The immunoblotting protein detection method based on microglial AD pathology according to claim 1, characterized in that , the chemiluminescent reagent is prepared in the S5, the treated blotting membrane is placed in the chemiluminescent reagent, and incubated for 1-2 minutes. After the incubation is completed, the excess chemiluminescent reagent on the edge or corner of the blotting membrane is absorbed, and the blotting membrane is placed flatly in a plastic bag so that the dry blotting membrane surface is in close contact with the film so that the chemiluminescent signal is transmitted to the film. The plastic bag containing the blotting membrane and the film is transferred to a dark room and exposed for 1-2 minutes in an environment without light interference. After the exposure is completed, the film is quickly taken out, and the film is scanned with a scanner or photographed with a camera. The acquired image is analyzed using a gel image processing device to measure the molecular weight and net optical density value of the target band.

Images