Name: A11-positive β-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis (Video) | JoVE Journal
Uploaded: 2018-05-22T13:30:00.000+00:00
Duration: 6 min 17 s
Description: Learn to prepare Aβ oligomers from synthetic peptide and assess them using dot blotting analysis. This simple, reliable method enables reproducible neuropharmacological research on Alzheimer's disease mechanisms.

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5 minutes

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 &amp; 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.

1. Solution Preparation
NOTE: See Table of Materials for reagent sources.
<ol>
	<li>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 &#176;C for up to 1 month.</li>
	<li>Prepare an anti-oligomer antibody A11 solution (1:1,000) by adding 10 &#181;L of antibody stock solution to 10 mL of the 5% BSA solution. Mix them completely by vortexing them. Store the solution at 4 &#176;C for up to 1 month.</li>
	<li>Prepare an anti-A&#946; antibody 6E10 solution (1:1,000) by adding 10 &#181;L of antibody stock solution to 10 mL of 5% BSA solution. Mix them completely by vortexing. Store the solution at 4 &#176;C for up to 1 month.</li>
	<li>Prepare an anti-fibrillar oligomer antibody OC solution (1:1,000) by adding 10 &#181;L of antibody stock solution to 10 mL of 5% BSA solution. Mix them completely by vortexing. Store the solution at 4 &#176;C for up to 1 month.</li>
	<li>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 &#176;C for up to 3 months.</li>
	<li>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.</li>
	<li>Prepare a secondary antibody solution by adding 10 &#181;L 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 &#176;C for up to 1 month.</li>
	<li>Dissolve 3.68 g of curcumin in 1 mL of dimethyl sulfoxide (DMSO) to form a 10-mM curcumin stock solution.</li>
	<li>Dissolve 5 mg of synthetic A&#946;1-42 in 2 mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to form a 2.5-mg/mL A&#946; monomer solution. Place it at room temperature (25 &#176;C) for 20 min. Make 100 &#181;L aliquots and store at -20 &#176;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.</li>
	<li>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.</li>
</ol>
2. Sample Preparation
NOTE: Perform the sample preparation 2 days before the dot blotting analysis.
<ol>
	<li>Add 900 &#181;L of double-distilled water to a tube of A&#946;1-42 monomer solution; the concentration of A&#946;1-42 will be 0.25 mg/mL. Place the mixture at room temperature for 20 min.</li>
	<li>Evaporate the solution with high-purity nitrogen gas until its volume is about 850 &#181;L. The concentration of the A&#946;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 &#181;L.</li>
	<li>Dilute the curcumin stock solution to a curcumin working solution (0.2 and 2 &#181;M) with double-distilled water. Mix the A&#946;1-42solution and curcumin working solution with a volume ratio of 1:1. The final concentrations of curcumin will be 0.1 and 1 &#181;M. 
	NOTE: Potential oligomer inhibitors can be mixed with the A&#946;1-42 solution in any ratio if needed.</li>
	<li>Shake the solution continuously in a magnetic agitator (see Table of Materials).
	<ol>
		<li>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.</li>
		<li>Shake the box at room temperature (25 &#176;C) for 48 h. 
		NOTE: The speed of the magnetic agitator is around 60 rpm.</li>
	</ol>
	</li>
	<li>Centrifuge the tubes for 15 min at 4 &#176;C and 18,000 x g. Collect the supernatant.</li>
</ol>
3. Dot Blotting Analysis
NOTE: All incubations are performed on a horizontal shaker.
<ol>
	<li>Cut nitrocellulose membrane into 1-cm wide strips. 
	NOTE: The width of the nitrocellulose membrane can be adjusted according to experimental needs.</li>
	<li>Place 2-&#181;L samples evenly on 2 strips (strip 1 and 2) with each point interval at 0.5 cm.</li>
	<li>Place the strips at room temperature for 5 min until the droplet on the band is dry.</li>
	<li>Incubate the strips with a 5% BSA solution for 30 min at room temperature.</li>
	<li>Rinse the strips with a TBST solution for 5 min at room temperature.</li>
	<li>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-A&#946; antibody 6E10 solution.</li>
	<li>Rinse the strips with a TBST solution three times, each time for 5 min at room temperature.</li>
	<li>Aspirate the TBST solution. Incubate the strips with a secondary antibody solution for 40 min at room temperature.</li>
	<li>Rinse the strips with a TBST solution three times, each time for 5 min at room temperature.</li>
	<li>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 &#181;L 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.</li>
	<li>Do a grayscale analysis using ImageJ (National Institutes of Health) or another image-processing software to obtain semi-quantitative results.</li>
</ol>

<ol><li>Lauren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+prion+protein+as+a+therapeutic+target+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Journal+of+Alzheimer%27s+Disease%22%5BJournal%5D)">Cellular prion protein as a therapeutic target in Alzheimer's disease.</a> Journal of Alzheimer's Disease. 38 (2), 227-244 (2014).</li>
<li>De Maio, A., Rivera, I., Cauvi, D. M., Arispe, N. <a target="_blank" href="http://www.cell.com/biophysj/comments/S0006-3495(16)33411-7">Modulation of amyloid peptide oligomerization and toxicity by extracellular Hsp70.</a> Biophysical Journal. 112 (3), 444(2017).</li>
<li>Di Scala, C., Chahinian, H., Yahi, N., Garmy, N., Fantini, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+Alzheimer%27s+beta-amyloid+peptides+with+cholesterol%3A+mechanistic+insights+into+amyloid+pore+formation%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Interaction of Alzheimer's beta-amyloid peptides with cholesterol: mechanistic insights into amyloid pore formation.</a> Biochemistry. 53 (28), 4489-4502 (2014).</li>
<li>Xiang, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fucoxanthin+inhibits+beta-amyloid+assembly+and+attenuates+beta-amyloid+oligomer-induced+cognitive+impairments%5BTitle%5D)+AND+%22Journal+of+Agricultural+and+Food+Chemistry%22%5BJournal%5D)">Fucoxanthin inhibits beta-amyloid assembly and attenuates beta-amyloid oligomer-induced cognitive impairments.</a> Journal of Agricultural and Food Chemistry. 65 (20), 4092-4102 (2017).</li>
<li>Chang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protection+against+beta-amyloid-induced+synaptic+and+memory+impairments+via+altering+beta-amyloid+assembly+by+bis%28heptyl%29-cognitin%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Protection against beta-amyloid-induced synaptic and memory impairments via altering beta-amyloid assembly by bis(heptyl)-cognitin.</a> Scientific Reports. 5, 10256(2015).</li>
<li>Yam, G. H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+amyloid+aggregate+forming+ability+of+TGFBI+mutants+that+cause+corneal+dystrophies%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">In vitro amyloid aggregate forming ability of TGFBI mutants that cause corneal dystrophies.</a> Investigative Ophthalmology & Visual Science. 53 (9), 5890-5898 (2012).</li>
<li>Tomaselli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+alpha-to-beta+conformational+transition+of+Alzheimer%27s+A+beta-%281-42%29+peptide+in+aqueous+media+is+reversible%3A+A+step+by+step+conformational+analysis+suggests+the+location+of+beta+conformation+seeding%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">The alpha-to-beta conformational transition of Alzheimer's A beta-(1-42) peptide in aqueous media is reversible: A step by step conformational analysis suggests the location of beta conformation seeding.</a> ChemBioChem. 7 (2), 257-267 (2006).</li>
<li>Shigemitsu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+magnetic+resonance+evidence+for+the+dimer+formation+of+beta+amyloid+peptide+1-42+in+1%2C1%2C1%2C3%2C3%2C3-hexafluoro-2-propanol%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1-42 in 1,1,1,3,3,3-hexafluoro-2-propanol.</a> Analytical Biochemistry. 498, 59-67 (2016).</li>
<li>Khan, M. V., Rabbani, G., Ahmad, E., Khan, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluoroalcohols-induced+modulation+and+amyloid+formation+in+conalbumin%5BTitle%5D)+AND+%22International+Journal+of+Biological+Macromolecules%22%5BJournal%5D)">Fluoroalcohols-induced modulation and amyloid formation in conalbumin.</a> International Journal of Biological Macromolecules. 70, 606-614 (2014).</li>
<li>Fang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((5-hydroxycyclopenicillone%2C+a+new+beta-amyloid+fibrillization+inhibitor+from+a+sponge-derived+fungus+trichoderma+sp+HPQJ-34%5BTitle%5D)+AND+%22Marine+Drugs%22%5BJournal%5D)">5-hydroxycyclopenicillone, a new beta-amyloid fibrillization inhibitor from a sponge-derived fungus trichoderma sp HPQJ-34.</a> Marine Drugs. 15 (8), 260(2017).</li>
<li>Shiao, Y. J., Su, M. H., Lin, H. C., Wu, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Echinacoside+ameliorates+the+memory+impairment+and+cholinergic+deficit+induced+by+amyloid+beta+peptides+via+the+inhibition+of+amyloid+deposition+and+toxicology%5BTitle%5D)+AND+%22Food+%26+Function%22%5BJournal%5D)">Echinacoside ameliorates the memory impairment and cholinergic deficit induced by amyloid beta peptides via the inhibition of amyloid deposition and toxicology.</a> Food & Function. 8 (6), 2283-2294 (2017).</li>
</ol>

The authors have nothing to disclose.

In this protocol, we have reported a method to prepare samples containing A&#946;1-42 oligomer, and to analyze the amounts of A11-positive A&#946;1-42 oligomer by a dot blotting analysis. Although our methods for the preparation of A&#946;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 A&#946;1-42 peptide. An aggregated A&#946;1-42 peptide can disassemb...

Alzheimer&#39;s disease (AD) is one of the most important neurodegenerative diseases affecting elderly people worldwide1. It is widely accepted that the abnormal aggregation of &#946;-amyloid (A&#946;) may be the leading pathological factor of AD. A&#946; aggregates are the main components of the senile plaques, one of the biological markers in the brains of AD patients. Moreover, A&#946; aggregates, including oligomers in particular, produce potent neurotoxicity, which might be the cause of neuronal death as AD progresses. Therefore, the inhibition of A&#946; oligomer formation might prevent neurodegeneration, and A&#946; oligomer inhibitors could be developed as disease-modifying treatments of AD. Many studies have used a synthetic A&#946; peptide to form oligomers in vitro, explore morphologies and structures of artificial A&#946; oligomers, and investigate the inhibitors of A&#946; oligomer using in vitro models2,3,4. However, different in vitro formation protocols of A&#946; 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 A&#946; oligomer is urgently needed.
Until now, not many methods have been reported to directly detect A&#946; 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 A&#946; oligomer in vitro5,6. For example, the morphology and structure of A&#946; oligomer can be observed in TEM. The relative amounts and molecular size of A&#946; aggregations could be measured by non-denaturing gel electrophoresis. ELISA could be used to determine A&#946; 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 A&#946; oligomer in different samples with the help of oligomer-specific and A&#946;-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 A&#946; oligomer inhibitors. The overall goal of this protocol is to describe a relatively simple, reliable, and reproducible method to prepare an A&#946;1-42 oligomer-rich sample, to analyze the amounts of A&#946;1-42 oligomer by dot blotting analysis, and to screen A&#946; oligomer inhibitors using semi-quantitative experimental results.

<table><tbody><tr><td>A11 (ab126892 Rb pAb to Amyloid Oligomers)</td><td>Abcam</td><td>GR91739-58</td><td></td></tr><tr><td>6E10 (beta-Amyloid Rabbit Ab)</td><td>Cell&amp;nbsp;Signalling Technology</td><td>2454S</td><td></td></tr><tr><td>OC (Anti-Amyloid Fibrils OC Antibody)</td><td>Millipore</td><td>AB2286</td><td></td></tr><tr><td>Horseradish Peroxidase (HRP) Marker Goat anti rabbit IgG (H+L)&amp;nbsp;</td><td>Beyotime</td><td>A0208</td><td></td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>GL Biochem</td><td>52487</td><td></td></tr><tr><td>1,1,1,3,3,3-Hexafiuoro-2-propanol</td><td>Aladdin</td><td>I1523078</td><td></td></tr><tr><td>Curcumin</td><td>Sigma</td><td>C1386</td><td></td></tr><tr><td>Albumin Bovine V</td><td>Solarbio</td><td>A8020</td><td></td></tr><tr><td>Sodium chloride</td><td>Sangon Biotech</td><td>D920BA0003</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>SCR</td><td>30166428</td><td></td></tr><tr><td>TRIS</td><td>Solarbio</td><td>T8060</td><td></td></tr><tr><td>Glycine</td><td>Solarbio</td><td>G8200</td><td></td></tr><tr><td>&amp;nbsp;Dimethyl sulfoxide</td><td>Solarbio</td><td>D8370</td><td></td></tr><tr><td>5&amp;times;nondenaturing gel PAGE Protein Marker</td><td>Beyotime</td><td>P0016</td><td></td></tr><tr><td>Genshare CFAS anyKD PAGE</td><td>Genshare</td><td>JC-PE022</td><td></td></tr><tr><td>Pure Nitrocellulose Blotting Membrane</td><td>Pall Corporation</td><td>T50189</td><td></td></tr><tr><td>Methanol</td><td>SCR</td><td>10014118</td><td></td></tr><tr><td>Ethanol</td><td>SCR</td><td>10009218</td><td></td></tr><tr><td>Super low range&amp;nbsp;protein Marker</td><td>Solarbio</td><td>PR1300</td><td></td></tr><tr><td>Transfer Membranes</td><td>Immobilon-PSQ</td><td>ISEQ00010</td><td></td></tr><tr><td>BeyoECL Star</td><td>Beyotime</td><td>P0018A</td><td></td></tr><tr><td>Commassie Blue Fast staining solution</td><td>Beyotime</td><td>P0017</td><td></td></tr><tr><td>All - automatic chemiluminescence imaging system</td><td>Tanon</td><td>Tanon 5200</td><td></td></tr><tr><td>Image J</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Image processing software</td><td>Adobe</td><td>Photoshop CS6</td><td></td></tr><tr><td>Magnetic agitator</td><td>Shanghai Huxi</td><td></td><td></td></tr></tbody></table>

a11-positive &#946;-amyloid oligomer preparation and assessment using dot blotting analysis

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental 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 oligomers after preparatio...

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

a11-positive-amyloid-oligomer-preparation-assessment-using-dot

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Learn to prepare Aβ oligomers from synthetic peptide and assess them using dot blotting analysis. This simple, reliable method enables reproducible neuropharmacological research on Alzheimer's disease mechanisms.

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

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