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 s...

<ol>
	<li>Lauren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+prion+protein+as+a+therapeutic+target+in+Alzheimer's+disease.">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.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+amyloid+peptide+oligomerization+and+toxicity+by+extracellular+Hsp70.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+Alzheimer's+beta-amyloid+peptides+with+cholesterol:+mechanistic+insights+into+amyloid+pore+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fucoxanthin+inhibits+beta-amyloid+assembly+and+attenuates+beta-amyloid+oligomer-induced+cognitive+impairments.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+beta-amyloid-induced+synaptic+and+memory+impairments+via+altering+beta-amyloid+assembly+by+bis(heptyl)-cognitin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+amyloid+aggregate+forming+ability+of+TGFBI+mutants+that+cause+corneal+dystrophies.">In vitro amyloid aggregate forming ability of TGFBI mutants that cause corneal dystrophies.</a> Investigative Ophthalmology &amp; Visual Science. 53 (9), 5890-5898 (2012).</li><li>Tomaselli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+evidence+for+the+dimer+formation+of+beta+amyloid+peptide+1-42+in+1,1,1,3,3,3-hexafluoro-2-propanol.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoroalcohols-induced+modulation+and+amyloid+formation+in+conalbumin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-hydroxycyclopenicillone,+a+new+beta-amyloid+fibrillization+inhibitor+from+a+sponge-derived+fungus+trichoderma+sp+HPQJ-34.">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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:1159px;" width="1158">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">A11 (ab126892 Rb pAb to Amyloid Oligomers)</td>
			<td style="width:219px;">Abcam</td>
			<td style="width:211px;">GR91739-58</td>
			<td style="width:359px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6E10 (beta-Amyloid Rabbit Ab)</td>
			<td>Cell&#160;Signalling Technology</td>
			<td>2454S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OC (Anti-Amyloid Fibrils OC Antibody)</td>
			<td>Millipore</td>
			<td>AB2286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horseradish Peroxidase (HRP) Marker Goat anti rabbit IgG (H+L)&#160;</td>
			<td>Beyotime</td>
			<td>A0208</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">A&#946;1-42</td>
			<td>GL Biochem</td>
			<td>52487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,1,1,3,3,3-Hexafiuoro-2-propanol</td>
			<td>Aladdin</td>
			<td>I1523078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curcumin</td>
			<td>Sigma</td>
			<td>C1386</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin Bovine V</td>
			<td>Solarbio</td>
			<td>A8020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>D920BA0003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium dodecyl sulfate</td>
			<td>SCR</td>
			<td>30166428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIS</td>
			<td>Solarbio</td>
			<td>T8060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Solarbio</td>
			<td>G8200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Dimethyl sulfoxide</td>
			<td>Solarbio</td>
			<td>D8370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5&#215;nondenaturing gel PAGE Protein Marker</td>
			<td>Beyotime</td>
			<td>P0016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Genshare CFAS anyKD PAGE</td>
			<td>Genshare</td>
			<td>JC-PE022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pure Nitrocellulose Blotting Membrane</td>
			<td>Pall Corporation</td>
			<td>T50189</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>SCR</td>
			<td>10014118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>SCR</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Super low range&#160;protein Marker</td>
			<td>Solarbio</td>
			<td>PR1300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer Membranes</td>
			<td>Immobilon-PSQ</td>
			<td>ISEQ00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BeyoECL Star</td>
			<td>Beyotime</td>
			<td>P0018A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Commassie Blue Fast staining solution</td>
			<td>Beyotime</td>
			<td>P0017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">All - automatic chemiluminescence imaging system</td>
			<td>Tanon</td>
			<td>Tanon 5200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image J</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image processing software</td>
			<td>Adobe</td>
			<td>Photoshop CS6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

&#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 A&#946;1-42 monomer can form an A&#946;1-42 oligomer after preparation, TEM analysis was used. No visible aggregates were observed in the HFIP-dissolved A&#946;1-42 monomer sample (Figure 1A). Moreover, mainly globular aggregates with a diameter of around 10 - 80 nm were observed in the A&#946;1-42 sample after 48 h of shaking, suggesting that A&#946;1-42 forms o...

Watch this Scientific Journal Video about A11-positive Î²-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis at JoVE.com

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

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.

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 ...

This method can help answer key questions in the neuropharmacological field. The main advantage of this technique is that it's simple, reliable, and reproducible to form beta-amyloid oligomer. We show demonstration of this method is critical as steps for preparing beta oligomer are difficult to learn.First, adjust the concentration of A-beta monomer solution to 0.25 milligrams per milliliter by adding 900 microliters of double-distilled water to the tube. Then continue evaporating the monomer solution with high purity nitrogen gas until the volume reaches about 850 microliters with a concentration of about 0.29 milligrams per milliliter. The presence of HR5P affects the formation of A-beta oligomer.Therefore, evaporating as much as possible is a critical step of this procedure. Next, use double-distilled water to dilute the curcumin stock solution to prepare a working solution of 0.2 and two micromolar. Maintain a ratio of one-to-one and mix the A-beta solution with curcumin to achieve the final concentration of curcumin at 0.1 and one micromolar.Then, leave the solution on a magnetic agitator. Attach a plastic divider box to the magnetic agitator. Position two magnetic stir bars at the two corners of the box, then place the tubes with the samples in the center of the box.Continue shaking the box at room temperature for 48 hours at 60 revolutions per minute, then centrifuge the tube at 18, 000 times G for 15 minutes at four degrees celsius. After centrifugation, collect the supernatant. For dot blotting analysis, take a nitrocellulose membrane and cut it into one-centimeter wide strips, then place two microliters of the sample evenly on the strips one and two with each point interval at 0.5 centimeters.When squirting the sample on membrane, it's necessary to spread evenly and the different droplets are controlled so that the different samples are not linked together. Next, incubate the strips at room temperature for five minutes for the droplets to dry out. After five minutes, incubate the strips with 5%bovine serum albumin for 30 minutes at room temperature.Once the incubation is over, wash the strips with TBST buffer for five minutes. Aspirate the buffer after the wash is done, then incubate strip one with anti-oligomer antibody A11 and strip two with anti-A-beta antibody 6E10 solution for an hour. After one hour, wash the strips with TBST for five minutes each three times, then aspirate the buffer and incubate the strips with secondary antibody solution for 40 minutes.After the secondary antibody incubation, again wash the strips with TBST for five minutes each three times. Then, add about 300 microliters of mixed ECL fluid to the surface of the moist strips, then expose the strips in an automatic chemiluminescence imaging system. TEM studies show the absence of any visible aggregates in the HFIP-dissolved A-beta monomer solution, however after 48 hours of incubation, the same monomer form oligomers, which are mainly globular aggregates with a diameter of 10 to 80 nanometers.On subjecting the A-beta monomer and oligomer-rich samples to dot blot analysis, it shows the presence of A11 positive A-beta oligomers in A-beta oligomer-rich samples. Interestingly, A-beta 6E10 positive A-beta peptides are present in both the A-beta monomer and oligomer-rich samples. Next, semi-quantitative analysis is also done to express the specific antibody-positive A-beta monomer and oligomer-rich samples as a percentage of control.A11 positive A-beta peptides are significantly higher in A-beta oligomer-rich samples in comparison to the monomers. On the contrary, A-beta 6E10 positive A-beta peptide is present in both the monomer and oligomer-rich samples. In fact, A-beta oligomers are also co-incubated with curcumin and A-beta oligomer inhibitor.Curcumin leads to significant reduction in the relative amount of A11 positive A-beta oligomer with decreased A11 staining in the curcumin co-incubated A-beta oligomer samples. However, varying concentrations of curcumin do not affect the number of A-beta peptides in the 6E10 positive monomer and oligomer-rich samples when compared to A11 positive samples. Once mastered, this technique can be done in several hours if it performed properly.After its development, this technique paved the way for researchers to explore drug candidates for neurodegenerative disorders such as Alzheimer's Disease. After watching this video, you should have a good understanding of how to prepare A-beta oligomer and to evaluate its amount.

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

Research

JoVE Journal

Biochemistry

11.6K visualizações.  Ningbo University. This method can help answer key questions in the neuropharmacological field. The main advantage of this technique is that it's simple, reliable, and reproducible to form beta-amyloid oligomer. We show demonstration of this method is critical as steps for preparing beta oligomer are difficult to learn.First, adjust the concentration of A-beta monomer solution to 0.25 milligrams per milliliter by adding 900 microliters of double-distilled water to the tube. Then continue evaporating the ...