A11-positive β-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

Abstract

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.

Calcium-Sensing Receptor Mediates β-Amyloid-Induced Synaptic Formation Impairment and Cognitive Deficits via Regulation of Cytosolic Phospholipase A2/Prostaglandin E2 Metabolic Pathway

“…A 50 μM Aβ 1–42 solution was obtained and stored at 4°C. The preparation is the combination of low molecular weight forms of soluble Aβ (Chunhui et al, 2018 ).…”

“…Then the collected supernatant was kept stirring for 48 h at room temperature. A 50 µM Aβ 1-42 solution was obtained and stored at 4 • C. The preparation is the combination of low molecular weight forms of soluble Aβ (Chunhui et al, 2018).…”

Dimeric Tacrine (10)-Hupyridone Effectively Combats Alzheimer’s Disease as A Multi-Target-Directed Ligand

“…Dot blotting analysis was also carried out according to the methods we reported previously [37]. Firstly, the mixture of equal volume of different concentrations of the drugs and Aβ solution were prepared.…”

Protocol for High-Throughput Screening of Neural Cell or Brain Tissue Protein Using a Dot-Blot Technique with Near-Infrared Imaging

“…Alternatively, dots can be applied freehand, as has been published by other laboratories (Au - Chunhui et al, 2018 , Tizon et al, 2010 ). In this case, the user should take care to keep the membrane as clean as possible (the infrared detection system is more sensitive to background contamination than other methods such as chemiluminescence ( Schutz-Geschwender et al, 2004 )).…”