Preparation and quality control method of A beta monomer
Through molecular sieve technology and immunoblotting analysis combined with thiosulfon T test, the problem of mixing oligomers and truncated peptides in Aβ monomers was solved, and high-purity Aβ monomers were prepared, which improved the accuracy of the research and the research effect of biological functions.
Patent Information
- Application Number
- CN202311762568.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-20
- Publication Date
- 2025-06-20
AI Technical Summary
The prior art is difficult to effectively avoid the problem of mixing Aβ oligomers and truncated peptides in Aβ monomers, which affects the aggregation kinetics and biological functions of Aβ monomers.
Molecular sieve technology combined with immunoblotting analysis and thiosulfon T test, Aβ monomers were isolated and identified, and quality control was carried out through antibody analysis of guanidine hydrochloride denaturation and different linear epitopes, and oligomers and truncated peptides were removed.
The high purity preparation of Aβ monomers is achieved, reducing the presence of oligomers and truncated peptides, and improving the accuracy of the aggregation kinetics and biological function research of Aβ monomers.
Smart Images
Figure CN120173082A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine and relates to a method for preparing and quality controlling Aβ monomers. Background Art
[0002] Alzheimer's disease (AD) is a common neurodegenerative disease and the main cause of senile dementia. Its clinical manifestations include progressive memory and cognitive dysfunction, etc., seriously affecting the quality of life of patients and their families. Amyloid plaques formed by abnormal aggregation of Aβ protein are one of the most prominent pathological features in the brains of AD patients. Aβ protein is a polypeptide containing 39-43 amino acids produced by proteolysis of amyloid precursor protein by β- and γ-secretases. The types of Aβ are Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43, among which Aβ42 has the greatest toxicity and is the main pathogenic factor of AD. A large number of basic and clinical studies have shown that Aβ plays an important role in the occurrence and development of AD. For example, the pathological deposition of Aβ is closely related to neurodegenerative lesions in AD, and the ratio of Aβ42 / 40 in the cerebrospinal fluid of patients increases significantly. Therefore, researchers generally use chemically synthesized Aβ as research materials to try to reveal its abnormal aggregation mechanism and study its various molecular and cellular functions in AD, and also widely use aggregated Aβ products as tools for drug screening and development.
[0003] In scientific research, researchers have found that Aβ in the brain tissue of AD patients exists in various different oligomeric forms, such as Aβ fibrils, Aβ spherical oligomers, dimers, etc. In order to more accurately reveal the process of generation of these oligomers and the factors affecting Aβ aggregation, high-quality Aβ monomers are indispensable research tools.
[0004] The most common way to prepare Aβ monomers is through chemical synthesis and liquid chromatography separation. To ensure the storage time of the synthesized polypeptide, freeze-drying is usually chosen to store the polypeptide powder. When used in experiments, it is directly dissolved in a small amount of DMSO and then diluted with the corresponding experimental buffer. The second method for preparing Aβ monomers is based on the freeze-dried powder of Aβ polypeptide prepared by chemical synthesis. After dissolving the Aβ polypeptide with hexafluoroisopropanol (HFIP) to disrupt the β-sheets in the Aβ freeze-dried powder, it is then dissolved with the help of DMSO and diluted with the experimental buffer. The third method is also based on the freeze-dried powder of Aβ polypeptide. The Aβ polypeptide is dissolved with sodium hydroxide (NaOH) and then diluted with the experimental buffer. The fourth method for preparing Aβ monomers is based on the freeze-dried powder of Aβ polypeptide prepared by synthesis. The Aβ freeze-dried powder is denatured overnight with 5 - 7 M guanidine hydrochloride (GuHCl), and then the Aβ monomers are obtained using molecular sieve technology according to the molecular weight of the Aβ monomers. Currently, the first three techniques all use strong bases or strong denaturing agents such as organic solvents to dissolve the Aβ freeze-dried powder in the corresponding experimental buffer. However, during the use process, Aβ oligomers still exist in the obtained Aβ solution, and these oligomers will affect the aggregation kinetics and biological functions of Aβ monomers. At the same time, there are still problems with the efficiency of amino acid condensation reactions in the current polypeptide chemical synthesis process. As the length of the polypeptide chain increases, the possibility of amino acid deletion becomes greater, and the possibility of errors in the deprotection reaction of amino acid side chains also becomes greater. Since the molecular weights of these incorrect polypeptides differ by less than 1 kd, it is very difficult to remove them during the liquid chromatography separation process. Therefore, the first three techniques also have the problem of being contaminated with truncated peptides. The fourth method uses guanidine hydrochloride denaturation and molecular sieve technology, which can remove the Aβ oligomers that exist due to incomplete denaturation, but there will still be the problem of being contaminated with truncated peptides.
[0005] In summary, how to avoid the contamination of Aβ monomers with Aβ oligomers and truncated peptides is one of the urgent problems to be solved in the field of Alzheimer's disease research. Summary of the Invention
[0006] In view of the deficiencies of the prior art and the actual needs, the present invention provides a method for preparing and quality controlling Aβ monomers. In order to better prepare Aβ monomers, the present invention combines a variety of protein purification and analysis techniques to solve the problem of contamination of Aβ monomers with Aβ oligomers and truncated peptides, and provides a method for preparing Aβ monomers and quality control.
[0007] To achieve the above object, the present invention adopts the following technical solutions:
[0008] In the first aspect, the present invention provides a method for preparing and quality controlling Aβ monomers. The method for preparing and identifying Aβ monomers includes the following steps:
[0009] (1) Mix the Aβ polypeptide with a buffer to obtain an Aβ polypeptide solution, and separate different components in the Aβ polypeptide solution according to the molecular weight of the polypeptide by using the molecular sieve method;
[0010] (2) Take the different components obtained in step (1) and perform immunoblot analysis. Use an antibody that recognizes the linear epitope of the Aβ polypeptide to quantitatively analyze the purity of Aβ monomers in each component;
[0011] (3) Use thioflavin T test to monitor the aggregation kinetic results of Aβ monomers in each component;
[0012] (4) According to the results of steps (2) and (3), perform quality control on the Aβ monomer component.
[0013] In the present invention, a method is designed to effectively separate Aβ monomers from synthetic Aβ polypeptides and perform quality control on the purity of the monomers. The molecular sieve technology is used to separate Aβ monomers to solve the problem that Aβ oligomers and truncated peptides are mixed in Aβ monomers. Combining immunoblot analysis (Western blot), antibody analysis techniques for different linear epitopes, and thioflavin T test (ThT assay) technology, perform purity analysis and quality control on the separated different components, and separate and analyze to prepare Aβ monomers with better purity.
[0014] In the present invention, the Aβ polypeptide refers to currently synthetic (such as chemical synthesis, etc.) Aβ polypeptides, which can be Aβ42, Aβ40, etc.
[0015] Preferably, step (1) specifically includes:
[0016] Mix the Aβ polypeptide with a buffer to obtain an Aβ polypeptide solution, inject the Aβ polypeptide solution into a purification column for elution, and separate different components in the Aβ polypeptide solution according to the molecular weight of the polypeptide based on the elution chromatogram peak.
[0017] Preferably, the buffer includes a guanidine hydrochloride solution.
[0018] Preferably, the concentration of the guanidine hydrochloride solution is 5 - 7 M (mol / L), including but not limited to 5.2 M, 5.5 M, 5.8 M, 6 M, 6.2 M, 6.5 M, or 6.8 M, etc.
[0019] Preferably, the ratio of the Aβ polypeptide to the buffer is 1 - 2 mg of Aβ polypeptide / mL of buffer, including but not limited to 1.2 mg of Aβ polypeptide / mL of buffer, 1.5 mg of Aβ polypeptide / mL of buffer, 1.6 mg of Aβ polypeptide / mL of buffer, 1.8 mg of Aβ polypeptide / mL of buffer, or 1.9 mg of Aβ polypeptide / mL of buffer.
[0020] Preferably, the temperature of the mixing is 20 to 35 °C, such as 21 °C, 22 °C, 25 °C, 28 °C, 29 °C, 30 °C, 32 °C or 34 °C, and the time is 10 to 16 h, including but not limited to 11 h, 12 h, 13 h, 14 h or 15 h.
[0021] Preferably, the packing material of the purification column includes Superdex75 increase and / or Superdex200 increase.
[0022] Preferably, the elution mobile phase includes ammonium bicarbonate solution.
[0023] Preferably, the concentration of the ammonium bicarbonate solution is 50 mM.
[0024] Preferably, the volume of the different components is 0.1 to 1 mL / mg based on the input amount of the Aβ polypeptide.
[0025] Preferably, step (2) specifically includes:
[0026] According to the absorption peak at 280 nm and the extinction coefficient of the Aβ polypeptide in the molecular sieve method process of step (1), calculate the concentration of the Aβ polypeptide in each component; according to the concentration of the Aβ polypeptide, in the immunoblot analysis, use a uniform loading amount; use an antibody that recognizes the linear epitope of the Aβ polypeptide to quantitatively analyze the purity of the Aβ monomer in each component.
[0027] Preferably, step (3) specifically includes:
[0028] According to the absorption peak at 280 nm and the extinction coefficient of the Aβ polypeptide in the molecular sieve method process of step (1), calculate the concentration of the Aβ polypeptide in each component; according to the concentration of the Aβ polypeptide, use thioflavin T test to monitor the aggregation kinetics results of the Aβ monomer in each component.
[0029] In the present invention, components having similar kinetics represent that these components contain the same ingredients.
[0030] Preferably, in the thioflavin T test, the working concentration of thioflavin T is 5 to 50 μM, including but not limited to 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 46 μM or 48 μM, and the working concentration of the Aβ polypeptide is 10 to 50 μM, including but not limited to 11 μM, 12 μM, 15 μM, 16 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 46 μM, 48 μM or 49 μM.
[0031] As a preferred technical solution, the method for preparing and quality controlling the Aβ monomer includes the following steps:
[0032] (1) Mix the Aβ polypeptide with a guanidine hydrochloride solution to obtain an Aβ polypeptide solution. Inject the Aβ polypeptide solution into a purification column for elution. According to the elution chromatographic peak, separate different components in the Aβ polypeptide solution according to the molecular weight of the polypeptide. The volume of the different components is 0.1 - 1 mL / mg based on the input amount of the Aβ polypeptide.
[0033] (2) Calculate the concentration of Aβ polypeptide in each component according to the absorption peak at 280 nm and the extinction coefficient of the Aβ polypeptide in the molecular sieve method process of step (1). According to the concentration of the Aβ polypeptide, take the different components obtained in step (1) and have a uniform loading amount in immunoblot analysis. Use an antibody that recognizes the linear epitope of the Aβ polypeptide to quantitatively analyze the purity of Aβ monomers in each component.
[0034] (3) According to the concentration of the Aβ polypeptide in step (2), take the different components obtained in step (1), and obtain the results of the aggregation kinetics of Aβ monomers in each component by using thioflavin T test monitoring.
[0035] (4) Perform quality control on the Aβ monomer component according to the results of steps (2) and (3).
[0036] Compared with the prior art, the present invention has the following beneficial effects:
[0037] In the present invention, the artificially synthesized Aβ polypeptide is denatured (such as using guanidine hydrochloride) and the molecular sieve technology is used to separate multi-components containing different Aβ from it. Through the immunoblotting technology of antibodies against different linear epitopes of Aβ, the Aβ monomer component after molecular sieve separation is further optimized to prepare Aβ monomers with better purity. At the same time, the aggregation kinetics of Aβ monomers is intervened by truncated peptides, and the Aβ monomer component after molecular sieve separation is further optimized by using ThT assay. Combining the results of immunoblotting and ThT assay, effective quality control of the purity of Aβ monomers is carried out, and Aβ42 monomer components with higher purity are identified, and better quality Aβ monomers can be obtained, which is beneficial to the research on the aggregation kinetics and biological functions of Aβ monomers in subsequent experiments. Description of the Drawings
[0038] Figure 1 It is a result diagram of separating Aβ42 monomers by molecular sieve technology;
[0039] Figure 2A It is a result diagram of identifying the purity of specific Aβ42 monomers in each component by immunoblotting method (antibody 3D6);
[0040] Figure 2B It is a result diagram of identifying the purity of specific Aβ42 monomers in each component by immunoblotting method (antibody 6E10);
[0041] Figure 2C The figure shows the purity results of specific Aβ42 monomers in each component identified by immunoblotting method (antibody 4G8);
[0042] Figure 2D The figure shows the purity results of specific Aβ42 monomers in each component identified by immunoblotting method (antibody 21F12);
[0043] Figure 3 The figure shows the purity results of Aβ42 monomers in each component judged by ThT aggregation kinetics monitoring; Detailed implementation mode
[0044] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments and drawings. It can be understood that the specific implementation mode described here is only used to explain the present invention, rather than limiting the present invention.
[0045] For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.
[0046] Example 1
[0047] This example uses Aβ42 protein as an example to verify the method for preparing and identifying Aβ monomers of the present invention.
[0048] The first part: Separating Aβ42 monomers using molecular sieve technology
[0049] (1) Denature 2 mg of synthetic Aβ42 powder (purchased from Qiangyao Biotech Co., Ltd., prepared by chemical synthesis method) with 1 mL of 7 M guanidine hydrochloride overnight at 25 °C.
[0050] (2) Combining molecular sieve technology, separate the denatured Aβ solution on a Superdex 75 increase purification column, with the mobile phase being 50 mM ammonium bicarbonate solution, pH = 8.5.
[0051] (3) The separation chromatogram is as shown in Figure 1 , GFS is a molecular sieve standard. Separate Aβ42 according to size using molecular sieve chromatography, collect one component every 0.5 mL, and obtain different components (Fraction, abbreviated as Fx),
[0052] The second part: Identifying the purity of the components of Aβ42 monomers using western blot and the technology of recognizing with different Aβ42 antibodies
[0053] (1) Calculate the protein concentration of each component containing Aβ42 according to the absorption peak at 280 nm during the molecular sieve experiment and the extinction coefficient of Aβ protein.
[0054] (2) According to the protein concentration of each component above, in the western blot experiment, use a uniform sample loading amount. For example, control the sample loading amount of each component at 20 ng.
[0055] (3) Use the current 4 antibodies that recognize different linear epitopes of Aβ42 (antibody 3D6 from creativebiolabs, catalog#TAB-0809CLV; antibody 6E10 from biolegend, catalog#SIG-39320; antibody 4G8 from biolegend, catalog#SIG-39200; and antibody 21F12 from Elan Pharmaceuticals, San Francisco, CA) to quantitatively analyze the content of specific Aβ42 protein in each component, and further identify the purity of Aβ42 in each component. The results are as Figures 2A - 2D shown. After comprehensive analysis with the four antibodies, the Aβ42 monomer components with relatively simple polypeptide compositions are Fx18, Fx19, Fx20, and Fx21.
[0056] Part III: Use the ThT assay to determine the purity of each component of Aβ42
[0057] Calculate the protein concentration of each component containing Aβ42 according to the absorption peak at 280 nm during the molecular sieve experiment and the extinction coefficient of Aβ42 protein.
[0058] According to the protein concentration of each component above, use the ThT assay to monitor the aggregation kinetics of each component protein. The ThT concentration is 20 μM, and the protein concentration is 20 μM.
[0059] According to the results of the aggregation kinetics, similar kinetics indicates that these components contain the same ingredients. The results are as Figure 3 shown. The aggregation curve patterns of Fx18, Fx19, and Fx20 are the same, indicating that they should have the same ingredients. Combining the results of western blotting and the THT assay, the Aβ42 monomer components prepared this time are determined, and the components with higher purity should be Fx18, Fx19, and Fx20.
[0060] Based on the experimental results of the second and third parts, perform quality control on the obtained Aβ42 monomers, select the Aβ42 monomer components with higher purity, and achieve the experimental purpose of further optimizing the preparation of Aβ42 monomers.
[0061] Example 2
[0062] In this example, the Aβ42 protein is taken as an example to verify the method for preparing and identifying Aβ monomers of the present invention.
[0063] Part 1: Separating Aβ42 monomers using molecular sieve technology
[0064] (1) Denature 3 mg of synthesized Aβ42 powder overnight at 20 °C using 1 mL of 5 M guanidine hydrochloride.
[0065] (2) Combine molecular sieve technology to separate the denatured Aβ solution on a Superdex 75 increase purification column, with the mobile phase being 50 mM ammonium bicarbonate solution, pH = 8.5.
[0066] (3) Separate Aβ42 by size using molecular sieve chromatography, collect one fraction every 1 mL, and obtain different fractions (Fraction, abbreviated as Fx).
[0067] Part 2: Identifying the purity of the components of Aβ42 monomers using western blot and the technology of recognizing with different Aβ42 antibodies
[0068] (1) Calculate the protein concentration of each fraction containing Aβ42 according to the absorption peak at 280 nm and the extinction coefficient of Aβ protein during the molecular sieve experiment.
[0069] (2) According to the protein concentration of each above-mentioned fraction, in the western blot experiment, use a uniform sample loading amount, for example, control the sample loading amount of each fraction at 20 ng.
[0070] (3) Use 4 current antibodies that recognize different linear epitopes of Aβ42 (antibody 3D6, antibody 6E10, antibody 4G8, and antibody 21F12) to quantitatively analyze the content of specific Aβ42 protein in each fraction, and further identify the purity of Aβ42 in each fraction. After comprehensive analysis with the four antibodies, the Aβ42 monomer fractions with relatively single polypeptide composition are Fx18, Fx19, Fx20, and Fx21.
[0071] Part 3: Using ThT assay to judge the purity of each fraction of Aβ42
[0072] Calculate the protein concentration of each fraction containing Aβ42 according to the absorption peak at 280 nm and the extinction coefficient of Aβ42 protein during the molecular sieve experiment.
[0073] According to the protein concentration of each above-mentioned fraction, use ThT assay to monitor the aggregation kinetics of each fraction of protein, with the ThT concentration being 5 μM and the protein concentration being 10 μM.
[0074] According to the aggregation kinetics results, similar kinetics indicates that these components contain the same ingredients. The aggregation curve patterns of Fx18, Fx19, and Fx20 are the same, suggesting they should have the same composition. Combining the Western blot and THT assay results, the composition of the prepared Aβ42 monomer is determined, and the components with higher purity should be Fx18, Fx19, and Fx20.
[0075] Based on the experimental results of the second and third parts, quality control was performed on the obtained Aβ42 monomer, and the Aβ42 monomer components with higher purity were selected, achieving the experimental purpose of further optimizing the preparation of Aβ42 monomer.
[0076] Example 3
[0077] In this example, the Aβ42 protein was used as an example to verify the method for preparing and identifying Aβ monomers of the present invention.
[0078] Part 1: Separating Aβ42 monomer using molecular sieve technology
[0079] (1) Denature 1 mg of synthesized Aβ42 powder overnight at 20 °C using 1 mL of 6 M guanidine hydrochloride.
[0080] (2) Combine molecular sieve technology to separate the denatured Aβ solution on a Superdex 200 increase purification column, with a mobile phase of 50 mM ammonium bicarbonate solution, pH = 8.5.
[0081] (3) Separate Aβ42 according to size using molecular sieve chromatography, collect one fraction every 0.1 mL, and obtain different fractions (Fraction, abbreviated as Fx).
[0082] Part 2: Identifying the purity of the components of Aβ42 monomer using Western blot and the technology of recognizing with different Aβ42 antibodies
[0083] (1) Calculate the protein concentration of each component containing Aβ42 based on the absorption peak at 280 nm and the extinction coefficient of Aβ protein during the molecular sieve experiment.
[0084] (2) According to the protein concentration of each above-mentioned component, in the Western blot experiment, use a uniform loading amount, for example, control the loading amount of each component at 15 ng.
[0085] (3) Using the current four antibodies that recognize different linear epitopes of Aβ42 (antibody 3D6 from Creative Biolabs, catalog #TAB-0809CLV; antibody 6E10 from Biolegend, catalog #SIG-39320; antibody 4G8 from Biolegend, catalog #SIG-39200; and antibody 21F12 from Elan Pharmaceuticals, San Francisco, CA), the content of specific Aβ42 protein in each component was quantitatively analyzed, and the purity of Aβ42 in each component was further identified. Through comprehensive analysis with the four antibodies, the Aβ42 monomer components with relatively simple polypeptide composition were Fx18, Fx19, Fx20, and Fx21.
[0086] Part III: Judging the purity of Aβ42 in each component using ThT assay
[0087] According to the absorption peak at 280 nm and the extinction coefficient of Aβ42 protein during the molecular sieve experiment, the protein concentration of each component containing Aβ42 was calculated.
[0088] According to the protein concentration of each component above, the aggregation kinetics of each component protein was monitored using ThT assay, with a ThT concentration of 50 μM and a protein concentration of 50 μM.
[0089] According to the aggregation kinetics results, similar kinetics indicates that these components contain the same ingredients. The aggregation curve patterns of Fx18, Fx19, and Fx20 are the same, so they should have the same ingredients. Combining the results of Western blot and THT assay, the components of the prepared Aβ42 monomer were determined, and the components with higher purity should be Fx18, Fx19, and Fx20.
[0090] Based on the experimental results of the second and third parts, quality control was carried out on the obtained Aβ42 monomer, and the Aβ42 monomer components with higher purity were selected, achieving the experimental purpose of further optimizing the preparation of Aβ42 monomer.
[0091] In summary, the present invention combines Western blot, antibody analysis technology for different linear epitopes, and ThT assay technology with the denaturing chemical synthesis of Aβ lyophilized powder such as guanidine hydrochloride and molecular sieve technology to separate Aβ monomers, and combines the results of Western blot and ThT assay to perform quality control on the purity of the obtained Aβ monomers, and analyzes and separates and prepares Aβ monomers with better purity.
[0092] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.
Claims
1. A method for preparing and quality controlling Aβ monomers, characterized in that, The preparation and identification method of the Aβ monomer comprises the following steps: (1) Mix the Aβ polypeptide with a buffer solution to obtain an Aβ polypeptide solution, and separate different components in the Aβ polypeptide solution according to the molecular weight of the polypeptide by a molecular sieve method; (2) Take the different components obtained in step (1), perform immunoblot analysis, and quantitatively analyze the purity of the Aβ monomer in each component using an antibody that recognizes the linear epitope of the Aβ polypeptide; (3) Monitor the aggregation kinetic results of the Aβ monomer in each component using thioflavin T test; (4) Perform quality control on the Aβ monomer component according to the results of step (2) and step (3).
2. The method for preparing and quality controlling Aβ monomers according to claim 1, characterized in that, Step (1) specifically includes: Mix the Aβ polypeptide with a buffer solution to obtain an Aβ polypeptide solution, inject the Aβ polypeptide solution into a purification column for elution, and separate different components in the Aβ polypeptide solution according to the molecular weight of the polypeptide based on the elution chromatogram peak.
3. The method for preparing and quality controlling Aβ monomers according to claim 2, characterized in that, The buffer solution includes a guanidine hydrochloride solution; Preferably, the concentration of the guanidine hydrochloride solution is 5-7 M.
4. The method for preparing and quality controlling Aβ monomers according to any one of claims 1 - 3, characterized in that, The ratio of the Aβ polypeptide to the buffer solution is 1-2 mg of Aβ polypeptide / mL of buffer solution.
5. The method for preparing and quality controlling Aβ monomers according to any one of claims 1 - 4, characterized in that, The temperature of the mixing is 20-35 °C, and the time is 10-16 h.
6. The method for preparing and quality controlling Aβ monomers according to claim 2, characterized in that, The packing material of the purification column includes Superdex75 increase and / or superdex 200 increase; Preferably, the mobile phase for elution includes an ammonium bicarbonate solution; Preferably, the volume of the different components is 0.1-1 mL / mg based on the input amount of the Aβ polypeptide.
7. The method for preparing and quality controlling Aβ monomers according to any one of claims 1 - 6, characterized in that, Step (2) specifically includes: Calculate the concentration of the Aβ polypeptide in each component according to the absorption peak at 280 nm and the extinction coefficient of the Aβ polypeptide during the molecular sieve method in step (1); according to the concentration of the Aβ polypeptide, use a uniform sample loading amount in the immunoblot analysis; quantitatively analyze the purity of the Aβ monomer in each component using an antibody that recognizes the linear epitope of the Aβ polypeptide.
8. The method for preparing and quality controlling Aβ monomers according to any one of claims 1 - 7, characterized in that, Step (3) specifically includes: Calculate the concentration of the Aβ polypeptide in each component according to the absorption peak at 280 nm and the extinction coefficient of the Aβ polypeptide during the molecular sieve method in step (1); according to the concentration of the Aβ polypeptide, monitor the aggregation kinetic results of the Aβ monomer in each component using thioflavin T test.
9. The method for preparing and quality controlling Aβ monomers according to claim 8, characterized in that, In the thioflavin T test, the working concentration of thioflavin T is 5-50 μM, and the working concentration of the Aβ polypeptide is 10-50 μM.
10. The method for preparing and quality controlling Aβ monomers according to any one of claims 1 - 9, characterized in that, The preparation and quality control method of the Aβ monomer comprises the following steps: (1) Mix the Aβ polypeptide with a guanidine hydrochloride solution to obtain an Aβ polypeptide solution, inject the Aβ polypeptide solution into a purification column for elution, and separate different components in the Aβ polypeptide solution according to the molecular weight of the polypeptide based on the elution chromatogram peak, and the volume of the different components is 0.1-1 mL / mg based on the input amount of the Aβ polypeptide; (2) Calculate the concentration of Aβ polypeptide in each component according to the absorption peak at 280 nm and the extinction coefficient of Aβ polypeptide in the molecular sieve method process of step (1); according to the concentration of the Aβ polypeptide, take different components obtained in step (1), and use a uniform sample loading amount in immunoblot analysis; use an antibody that recognizes the linear epitope of Aβ polypeptide to quantitatively analyze the purity of Aβ monomers in each component; (3) According to the concentration of the Aβ polypeptide described in step (2), take different components obtained in step (1), and obtain the results of monitoring the aggregation kinetics of Aβ monomers in each component using thioflavin T testing; (4) Perform quality control on the Aβ monomer component according to the results of steps (2) and (3).