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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

1. Solution Preparation

NOTE: See Table of Materials for reagent sources.

1. 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.
2. 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 solution at 4 °C for up to 1 month.
3. Prepare an anti-Aβ antibody 6E10 solution (1:1,000) by adding 10 µL of antibody stock solution to 10 mL of 5% BSA solution. Mix them completely by vortexing. Store the solution at 4 °C for up to 1 month.
4. Prepare an anti-fibrillar oligomer antibody OC solution (1:1,000) by adding 10 µL of antibody stock solution to 10 mL of 5% BSA solution. Mix them completely by vortexing. Store the solution at 4 °C for up to 1 month.
5. Prepare a Tris-buffered saline (TBS) stock solution by adding 24 g of Tris base and 88 g of NaCl to 1,000 mL of double-distilled water. Adjust the pH to 7.4. Store the solution at 4 °C for up to 3 months.
6. Prepare a Tris-buffered saline and Tween-20 (TBST) solution by adding 1 mL of Tween-20 to 100 mL of TBS stock solution and 900 mL double-distilled water.
7. Prepare a secondary antibody solution by adding 10 µL of horseradish peroxidase (HRP) Goat anti-Rabbit IgG (H + L) to 10 mL of TBST solution. Mix them completely by vortexing them. Store the solution at 4 °C for up to 1 month.
8. Dissolve 3.68 g of curcumin in 1 mL of dimethyl sulfoxide (DMSO) to form a 10-mM curcumin stock solution.
9. Dissolve 5 mg of synthetic Aβ_1-42 in 2 mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to form a 2.5-mg/mL Aβ monomer solution. Place it at room temperature (25 °C) for 20 min. Make 100 µL aliquots and store at -20 °C for up to 6 months.
   NOTE: This procedure should be run as fast as possible. The pipette tips should be cut off to ensure accurate pipetting.
10. Prepare electrochemiluminescence (ECL) fluid by mixing ECL fluid A and B with a volume ratio of 1:1. This solution should be prepared just before use.

2. Sample Preparation

NOTE: Perform the sample preparation 2 days before the dot blotting analysis.

1. Add 900 µL of double-distilled water to a tube of Aβ_1-42 monomer solution; the concentration of Aβ_1-42 will be 0.25 mg/mL. Place the mixture at room temperature for 20 min.
2. Evaporate the solution with high-purity nitrogen gas until its volume is about 850 µL. The concentration of the Aβ_1-42 solution will be about 0.29 mg/mL.
   NOTE: The solution should be shaken from time to time to ensure the HFIP is fully evaporated. Normally, after 30 min of evaporation, the volume of the residual solution is about 850 µL.
3. Dilute the curcumin stock solution to a curcumin working solution (0.2 and 2 µM) with double-distilled water. Mix the Aβ_1-42solution and curcumin working solution with a volume ratio of 1:1. The final concentrations of curcumin will be 0.1 and 1 µM.
   NOTE: Potential oligomer inhibitors can be mixed with the Aβ_1-42 solution in any ratio if needed.
4. Shake the solution continuously in a magnetic agitator (see Table of Materials).
   1. Fix a plastic divider box to the magnetic agitator. Place 2 magnetic stir bars at 2 corners of the box and place the sample tubes in the center of the box.
   2. Shake the box at room temperature (25 °C) for 48 h.
      NOTE: The speed of the magnetic agitator is around 60 rpm.
5. Centrifuge the tubes for 15 min at 4 °C and 18,000 x g. Collect the supernatant.

3. Dot Blotting Analysis

NOTE: All incubations are performed on a horizontal shaker.

1. Cut nitrocellulose membrane into 1-cm wide strips.
   NOTE: The width of the nitrocellulose membrane can be adjusted according to experimental needs.
2. Place 2-µL samples evenly on 2 strips (strip 1 and 2) with each point interval at 0.5 cm.
3. Place the strips at room temperature for 5 min until the droplet on the band is dry.
4. Incubate the strips with a 5% BSA solution for 30 min at room temperature.
5. Rinse the strips with a TBST solution for 5 min at room temperature.
6. Aspirate the TBST solution. For 1 h at room temperature, incubate strip 1 with an anti-oligomer antibody A11 solution and strip 2 with an anti-Aβ antibody 6E10 solution.
7. Rinse the strips with a TBST solution three times, each time for 5 min at room temperature.
8. Aspirate the TBST solution. Incubate the strips with a secondary antibody solution for 40 min at room temperature.
9. Rinse the strips with a TBST solution three times, each time for 5 min at room temperature.
10. Evenly apply the mixed ECL fluid to the surface of the strips. Expose the strips in an automatic chemiluminescence imaging system (see Table of Materials).
    NOTE: Normally, 300 µL of ECL fluid is enough for one membrane. The membrane must be kept moist during the exposure. The exposure time is automatically calculated by the imaging system.
11. Do a grayscale analysis using ImageJ (National Institutes of Health) or another image-processing software to obtain semi-quantitative results.

Wyniki

To investigate whether an Aβ_1-42 monomer can form an Aβ_1-42 oligomer after preparation, TEM analysis was used. No visible aggregates were observed in the HFIP-dissolved Aβ_1-42 monomer sample (Figure 1A). Moreover, mainly globular aggregates with a diameter of around 10 - 80 nm were observed in the Aβ_1-42 sample after 48 h of shaking, suggesting that Aβ_1-42 forms o...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
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