This protocol describes how to prepare Aβ oligomers from a synthetic peptide in vitro and to evaluate relative amounts of Aβ oligomer by a dot blotting analysis.
β-amyloid (Aβ) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, Aβ oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer's disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, Aβ oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of Aβ oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen Aβ1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic Aβ1-42 oligomer with anti-parallel β-sheet structures. In this protocol, we describe how to prepare an A11-positive Aβ1-42 oligomer-rich sample from a synthetic Aβ1-42 peptide in vitro and to evaluate relative amounts of A11-positive Aβ1-42 oligomer in samples by a dot blotting analysis using A11 and Aβ1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive Aβ1-42 oligomer can also be screened from semi-quantitative experimental results.
Alzheimer's disease (AD) is one of the most important neurodegenerative diseases affecting elderly people worldwide1. It is widely accepted that the abnormal aggregation of β-amyloid (Aβ) may be the leading pathological factor of AD. Aβ aggregates are the main components of the senile plaques, one of the biological markers in the brains of AD patients. Moreover, Aβ aggregates, including oligomers in particular, produce potent neurotoxicity, which might be the cause of neuronal death as AD progresses. Therefore, the inhibition of Aβ oligomer formation might prevent neurodegeneration, and Aβ oligomer inhibitors could be developed as disease-modifying treatments of AD. Many studies have used a synthetic Aβ peptide to form oligomers in vitro, explore morphologies and structures of artificial Aβ oligomers, and investigate the inhibitors of Aβ oligomer using in vitro models2,3,4. However, different in vitro formation protocols of Aβ oligomer could lead to oligomer with different morphological characteristics, which might cause the incomparable results among different research groups. Therefore, a standard formation protocol for Aβ oligomer is urgently needed.
Until now, not many methods have been reported to directly detect Aβ oligomers. Transmission electronic microscopy (TEM), non-denaturing gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and dot blotting analysis can be used to examine the amount and/or morphology of Aβ oligomer in vitro5,6. For example, the morphology and structure of Aβ oligomer can be observed in TEM. The relative amounts and molecular size of Aβ aggregations could be measured by non-denaturing gel electrophoresis. ELISA could be used to determine Aβ oligomer in serum, plasma, and extracts from brain tissue. Lastly, dot blotting analysis, a technique used for detecting, analyzing, and identifying proteins, could be used to evaluate the relative concentration of Aβ oligomer in different samples with the help of oligomer-specific and Aβ-specific antibodies. Moreover, a dot blotting assay offers significant time savings, as gel electrophoresis and the blotting procedures for gels are not required. Therefore, this assay is normally used to screen potential Aβ oligomer inhibitors. The overall goal of this protocol is to describe a relatively simple, reliable, and reproducible method to prepare an Aβ1-42 oligomer-rich sample, to analyze the amounts of Aβ1-42 oligomer by dot blotting analysis, and to screen Aβ oligomer inhibitors using semi-quantitative experimental results.
In this protocol, we have reported a method to prepare samples containing Aβ1-42 oligomer, and to analyze the amounts of A11-positive Aβ1-42 oligomer by a dot blotting analysis. Although our methods for the preparation of Aβ1-42 oligomer-rich samples are quite simple, reliable, and reproducible, there are still some points to be noticed. Firstly, HFIP is used to dissolve the synthetic Aβ1-42 peptide. An aggregated Aβ1-42 peptide can disassemb…
The authors have nothing to disclose.
This work was supported by grants from the National Natural Science Foundation of China (U1503223, 81673407, 21475131), the Applied Research Project on Nonprofit Technology of Zhejiang Province (2016C37110), the Ningbo International Science and Technology Cooperation Project (2014D10019), the Ningbo Municipal Innovation Team of Life Science and Health (2015C110026), the Ningbo Sci & Tech Project for Common Wealth (2017C50042), the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund, and the K. C. Wong Magna Fund at Ningbo University.
A11 (ab126892 Rb pAb to Amyloid Oligomers) | Abcam | GR91739-58 | |
6E10 (beta-Amyloid Rabbit Ab) | Cell Signalling Technology | 2454S | |
OC (Anti-Amyloid Fibrils OC Antibody) | Millipore | AB2286 | |
Horseradish Peroxidase (HRP) Marker Goat anti rabbit IgG (H+L) | Beyotime | A0208 | |
Aβ1-42 | GL Biochem | 52487 | |
1,1,1,3,3,3-Hexafiuoro-2-propanol | Aladdin | I1523078 | |
Curcumin | Sigma | C1386 | |
Albumin Bovine V | Solarbio | A8020 | |
Sodium chloride | Sangon Biotech | D920BA0003 | |
Sodium dodecyl sulfate | SCR | 30166428 | |
TRIS | Solarbio | T8060 | |
Glycine | Solarbio | G8200 | |
Dimethyl sulfoxide | Solarbio | D8370 | |
5×nondenaturing gel PAGE Protein Marker | Beyotime | P0016 | |
Genshare CFAS anyKD PAGE | Genshare | JC-PE022 | |
Pure Nitrocellulose Blotting Membrane | Pall Corporation | T50189 | |
Methanol | SCR | 10014118 | |
Ethanol | SCR | 10009218 | |
Super low range protein Marker | Solarbio | PR1300 | |
Transfer Membranes | Immobilon-PSQ | ISEQ00010 | |
BeyoECL Star | Beyotime | P0018A | |
Commassie Blue Fast staining solution | Beyotime | P0017 | |
All – automatic chemiluminescence imaging system | Tanon | Tanon 5200 | |
Image J | National Institutes of Health | ||
Image processing software | Adobe | Photoshop CS6 | |
Magnetic agitator | Shanghai Huxi |