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

Summary

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.

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 Aβ_1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic Aβ_1-42 oligomer with anti-parallel β-sheet structures. In this protocol, we describe how to prepare an A11-positive Aβ_1-42 oligomer-rich sample from a synthetic Aβ_1-42 peptide in vitro and to evaluate relative amounts of A11-positive Aβ_1-42 oligomer in samples by a dot blotting analysis using A11 and Aβ_1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive Aβ_1-42 oligomer can also be screened from semi-quantitative experimental results.

Introduction

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 models^2,3,4. 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 vitro^5,6. 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 Aβ_1-42 oligomer-rich sample, to analyze the amounts of Aβ_1-42 oligomer by dot blotting analysis, and to screen Aβ oligomer inhibitors using semi-quantitative experimental results.

Protocol

1. Solution Preparation NOTE: See Table of Materials for reagent sources. Prepare a 5% bovine serum albumin (BSA) solution by adding 5 g of BSA to 100 mL of double-distilled water. Mix them completely by vortexing them. Store the solution at 4 °C for up to 1 month. Prepare an anti-oligomer antibody A11 solution (1:1,000) by adding 10 µL of antibody stock solution to 10 mL of the 5% BSA solution. Mix them completely by vortexing them. Store the s…

Representative Results

To investigate whether an Aβ1-42 monomer can form an Aβ1-42 oligomer after preparation, TEM analysis was used. No visible aggregates were observed in the HFIP-dissolved Aβ1-42 monomer sample (Figure 1A). Moreover, mainly globular aggregates with a diameter of around 10 – 80 nm were observed in the Aβ1-42 sample after 48 h of shaking, suggesting that Aβ1-42 forms o…

Discussion

In this protocol, we have reported a method to prepare samples containing Aβ_1-42 oligomer, and to analyze the amounts of A11-positive Aβ_1-42 oligomer by a dot blotting analysis. Although our methods for the preparation of Aβ_1-42 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 Aβ_1-42 peptide. An aggregated Aβ_1-42 peptide can disassemb…

Materials

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