Name: a11-positive &#946;-amyloid oligomer preparation and assessment using dot blotting analysis
Uploaded: 2018-05-22T13:30:00
Description: Watch this Scientific Journal Video about A11-positive ÃŽÂ²-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis at JoVE.com

Dette arbejde blev støttet af tilskud fra National Natural Science Foundation of China (U1503223, 81673407, 21475131), forskningsprojektet anvendes på Nonprofit teknologi af Zhejiang provinsen (2016C 37110), Ningbo International videnskab og teknologi Samarbejdsprojekt (2014D 10019), Ningbo Municipal Innovation Team af biovidenskab og sundhed (2015C 110026), Ningbo Sci & Tech projekt for fælles rigdom (2017C 50042), Li Dak summen Yip Yio hage Kenneth Li Marine biofarmaceutiske Udviklingsfond, og K. C. Wong Magna fonden på Ningbo Universitet.

1. løsning forberedelse
Bemærk: Se Tabel af materialer for reagens kilder.
<ol><li>Forbered en 5% bovint serumalbumin (BSA) løsning ved at tilføje BSA. 5 g i 100 mL dobbeltdestilleret vand. Bland dem helt af vortexing dem. Gemme løsning ved 4 ° C i op til 1 måned.</li>
	<li>Forbered en anti-oligomer antistof A11 løsning (1:1, 000) ved at tilføje 10 µL antistof stamopløsning 10 ml 5% BSA løsning. Bland dem helt af vortexing dem. Gemme løsning ved 4 ° C i op til ...

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echinacoside+ameliorates+the+memory+impairment+and+cholinergic+deficit+induced+by+amyloid+beta+peptides+via+the+inhibition+of+amyloid+deposition+and+toxicology.">Echinacoside ameliorates the memory impairment and cholinergic deficit induced by amyloid beta peptides via the inhibition of amyloid deposition and toxicology.</a> Food &amp; Function. 8 (6), 2283-2294 (2017).</li></ol>

I denne protokol, har vi rapporteret en metode til at forberede prøverne der indeholder Aβ1-42 oligomer og analysere mængder af A11-positive Aβ1-42 oligomer af en prik blotting analyse. Selv om vores metoder til forberedelse af Aβ1-42 oligomer-rige prøver er ganske enkel, pålidelig og reproducerbar, er der stadig nogle punkter at blive bemærket. For det første der HFIP bruges til at opløse den syntetiske Aβ1-42 peptid. En aggregeret Aβ1-42 peptid kan adsk...

Alzheimers sygdom (AD) er en af de vigtigste neurodegenerative sygdomme hos ældre mennesker over hele verden1. Det er almindeligt accepteret, at unormale sammenlægning af β-amyloid (Aβ) kan være den førende patologiske faktor af annonce. Aβ aggregater er de vigtigste komponenter i de senile plaques, en af de biologiske markører i hjernen hos AD patienter. Derudover producere Aβ aggregater, herunder oligomerer især potent neurotoksicitet, som kan være årsag til neuronal død som AD skrider frem. Derfor, hæmning af Aβ oligomer dannelse kan forhindre neurodegeneration, og Aβ oligomer hæmmere kan udvikles som Sygdomsmodificerende behandlinger af annonce. Mange undersøgelser har brugt en syntetisk Aβ peptid at danne oligomerer in vitro, udforske morfologier og strukturer af kunstige Aβ oligomerer og undersøge hæmmere af Aβ oligomer ved hjælp af in vitro- modeller2,3 , 4. imidlertid forskellige in vitro- dannelsen protokoller af Aβ oligomer kunne føre til oligomer med forskellige morfologiske karakteristika, der kan forårsage de uforlignelige resultater blandt forskellige forskergrupper. Derfor er en standard dannelse protokol for Aβ oligomer bydende nødvendigt.
Indtil nu, er ikke mange metoder blevet rapporteret til direkte registrere Aβ oligomerer. Transmission elektroniske mikroskopi (TEM), ikke-denatureringen gelelektroforese, enzymmaerket assay (ELISA) og dot blotting analyse kan bruges til at undersøge beløb og/eller morfologi af Aβ oligomer in vitro-5, 6. For eksempel morfologi og struktur af Aβ oligomer kan observeres i TEM. De relative mængder og molekylær størrelse af Aβ sammenlægninger kunne måles af ikke-denatureringen gelelektroforese. ELISA kan bruges til at bestemme Aβ oligomer i serum, plasma og uddrag af hjernevæv. Dot blotting analyse, en teknik, der anvendes til påvisning af, kan analysere og identificere proteiner, endelig anvendes til at evaluere den relative koncentration af Aβ oligomer i forskellige prøver ved hjælp af oligomer-specifikke og Aβ-specifikke antistoffer. Desuden tilbyder en prik blotting assay betydelige tidsbesparelser, som gelelektroforese og blotting procedurerne for geler ikke er påkrævet. Derfor, denne analyse bruges normalt til at skærmen potentielle Aβ oligomer hæmmere. Det overordnede mål med denne protokol er at beskrive en relativt simpel, pålidelig og reproducerbar metode for at forberede en Aβ1-42 oligomer-rige prøven, til at analysere mængden af Aβ1-42 oligomer af dot blotting analyse, og at skærmen Aβ oligomer inhibitorer ved hjælp af semi-kvantitative eksperimentelle resultater.

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	<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

Β-amyloid (Aβ) er en hydrofobe peptid med en iboende tendens til at selv samle til aggregater. Blandt forskellige aggregater, Aβ oligomer er bredt accepteret som den førende nervegift i forløbet af Alzheimers sygdom (AD) og er anset for at være den afgørende begivenhed i patogenesen af annonce. Derfor Aβ oligomer hæmmere kan forhindre neurodegeneration og har potentiale til at blive udviklet som Sygdomsmodificerende behandlinger af annonce. Men forskellige dannelse protokoller af Aβ oligomer kan føre til oligomerer med forskellige karakteristika. Desuden er der ikke mange metoder til effektivt skærmen Aβ1-42 oligomer hæmmere. En A11 antistof kan reagere med et undersæt af giftige Aβ1-42 oligomer med anti-parallelle β-plade strukturer. I denne protokol beskriver vi, hvordan at forberede en A11-positive Aβ1-42 oligomer-rige prøve fra en syntetisk Aβ1-42 peptid i vitro og evaluere relative mængder af A11-positive Aβ1-42 oligomer i prøver af en prik blotting analyse ved hjælp af A11 og Aβ1-42-specifikke 6E10 antistoffer. Ved hjælp af denne protokol, kan hæmmere af A11-positive Aβ1-42 oligomer også blive screenet fra semi-kvantitative eksperimentelle resultater.

For at undersøge, om en Aβ1-42 monomer kan danne en Aβ1-42 oligomer efter tilberedning, blev TEM analyse anvendt. Ingen synlige aggregater blev observeret i HFIP-opløst Aβ1-42 monomer prøve (fig. 1A). Derudover hovedsageligt kugleformede aggregater med en diameter på omkring 10-80 nm blev observeret i Aβ1-42 prøve efter 48 h, rysten, tyder på, at Aβ1-42 danner oligomerer efter ...

Watch this Scientific Journal Video about A11-positive ÃŽÂ²-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis at JoVE.com

A11-positive ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

Denne protokol beskriver, hvordan at forberede Aβ oligomerer fra en syntetisk peptid i vitro og evaluere relative mængder af Aβ oligomer af en prik blotting analyse.

A11-positive β-amyloid Oligomer forberedelse og vurdering ved hjælp af Dot Blotting analyse

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

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

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