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

这项工作得到了中国国家自然科学基金 (U1503223, 81673407, 21475131) 的资助, 浙江省非营利技术应用研究项目 (2016C37110), 宁波国际科技合作项目 (2014D10019)、宁波市生命科学与健康创新团队 (2015C110026)、宁波市科技 & 科技项目共同财富 (2017C50042)、李家和业杨厝港钦利海洋生物制药发展基金,宁波大学的肯塔基州金麦格纳基金。

1. 解决方案准备
注: 有关试剂来源, 请参阅材料表。
<ol><li>在100毫升的双蒸馏水中加入5克 bsa, 制备5% 牛血清白蛋白 (bsa) 溶液。涡流他们完全混合。将解决方案存储在摄氏4摄氏度, 最多1月。</li>
	<li>在 5% BSA 溶液的10毫升中加入10µL 抗体储存液, 制备抗低聚物抗体 A11 溶液 (1:1, 000)。涡流他们完全混合。将解决方案存储在摄氏4摄氏度, 最多1月。</li>
	<li>在10毫升 5% BSA...

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在本协议中, 我们报告了一种方法, 用于准备包含 Aβ1-42低聚物的样本, 并通过斑点印迹分析来分析 A11-positive Aβ1-42寡聚的数量。虽然我们的制备 Aβ1-42富聚物样品的方法非常简单、可靠、重现性好, 但仍有一些值得注意的点。首先, HFIP 用于溶解合成 Aβ1-42肽。聚合 Aβ1-42肽可以在 HFIP 溶液中分解成单体。但是, HFIP 容易挥发, HFIP 的粘度很低。因此, Aβ1-42</s...

阿尔茨海默病 (AD) 是影响全球老年人的最重要的神经退行性疾病之一1。普遍认为β淀粉样蛋白 (Aβ) 的异常聚集可能是 AD 的主要病理因子。Aβ骨料是老年斑块的主要成分, 是 AD 患者大脑中的生物学标志物之一。此外, Aβ聚集物, 特别是寡聚物, 产生强大的神经毒性, 这可能是导致神经元死亡的原因。因此, 抑制 Aβ寡聚物的形成可以防止变性, Aβ寡聚物抑制剂可作为 AD 的病害修饰处理。许多研究使用合成 Aβ肽在体外形成寡聚物, 探讨人工 Aβ寡聚的形貌和结构, 并利用体外模型2,3对 Aβ寡聚物的抑制剂进行研究。,4. 然而, 不同的体外形成协议的 Aβ寡聚物可能导致不同形态特征的聚类, 这可能导致不同研究组之间的不可比拟的结果。因此, 迫切需要一种标准的 Aβ齐聚物生成协议。
到目前为止, 还没有多少方法可以直接检测 Aβ寡聚物。透射电镜 (TEM)、非变性凝胶电泳、酶联免疫吸附试验 (ELISA) 和斑点印迹分析可用于检测 Aβ寡聚物的数量和/或形态学体外5,6. 例如, Aβ齐聚物的形态和结构可以在 TEM 中观察到。用非变性凝胶电泳法测定 Aβ聚合的相对含量和分子量。ELISA 可用于测定血清、血浆和脑组织提取物中的 Aβ寡聚体。最后, 斑点印迹分析, 一种用于检测、分析和识别蛋白质的技术, 可用于评价不同样品中 Aβ齐聚物的相对浓度, 并借助于寡聚特异性和 Aβ特异抗体。此外, 斑点印迹检测提供了大量的时间节省, 因为凝胶电泳和印迹程序的凝胶是不需要的。因此, 这种检测通常用于筛查潜在的 Aβ寡聚物抑制剂。本协议的总体目标是描述一种相对简单、可靠且可重现的方法, 用于准备 Aβ1-42富聚物的样本, 通过斑点印迹分析来分析 Aβ1-42低聚物的数量, 并筛选 Aβ寡聚使用半定量实验结果的抑制剂。

<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

β淀粉样蛋白 (Aβ) 是一种疏水性肽, 具有自组装成骨料的内在倾向。在各种团聚体中, Aβ寡聚物作为阿尔茨海默病 (ad) 进展的主要神经毒素被广泛接受, 被认为是 ad 发病的关键事件。因此, Aβ寡聚物抑制剂可以防止变性, 并有潜力发展为疾病修改治疗的 AD。然而, Aβ寡聚物的不同形成方案可能会导致不同特征的寡聚。此外, 有效筛选 Aβ1-42寡聚物抑制剂的方法也不多。A11 抗体可以与含有反平行β片结构的毒性 Aβ1-42齐聚物的子集发生反应。在本协议中, 我们描述了如何从 Aβ的合成 Aβ1-42肽的体外准备一个 A11-positive 的1-42富齐聚的样本, 并通过斑点印迹来评估样本中 A11-positive Aβ1-42齐聚物的相对数量使用 A11 和 Aβ1-42特定6E10 抗体进行分析。使用该协议, A11-positive Aβ1-42齐聚物的抑制剂也可以从半定量实验结果中筛选出来。

为研究 Aβ1-42单体在制备后是否能形成 Aβ1-42齐聚物, 采用 TEM 分析。在 HFIP 溶解 Aβ1-42单体样本中未观察到可见骨料 (图 1A)。此外, 在48小时的震动后, 在 Aβ1-42样本中观察到了直径约 10-80 nm 的球状骨料, 这表明 Aβ1-42在准备后形成寡聚 (图 1B)。
<p class="jove_content...

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

本协议描述如何从合成肽体外制备 Aβ寡聚物, 并通过斑点印迹分析来评价 Aβ的相对含量。

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

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