Name: protein digestion, ultrafiltration, and size exclusion chromatography to optimize the isolation of exosomes from human blood plasma and serum
Uploaded: 2018-04-13T15:00:00
Description: Watch this Scientific Journal Video about Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum at JoVE.com

这项研究得到了来自 CSU (KMD)、ATCC 合同 #2016 0550-0002 (NIAID HHSN272201600013C 分包)、比尔和梅琳达盖茨基金会 (OPP1039688) (KMD/NKG) 的内部资金的支持。我们感谢美国国家科学基金会在科罗拉多州立大学 (拉吴) 暑期项目的研究经验, 以得到更多的支持。

这项工作在科罗拉多州立大学的机构审查委员会 (IRB) 初步审查后被确定为非人类专题研究, 因为所有的人类样本都是从 Bioreclamation IVT biorepository 中提取出来的样本, 并根据 IRB 批准的协议收集。
1. 通过造粒较大的囊泡和细胞碎片制备粗样品 (血浆或血清)
注意:安全考虑: 血浆、血清和其他生物液体是 biohazardous 材料, 必须特别小心处理, 同时佩戴?...

<ol>
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从血液中提高外来体纯度和产量的优化方法, 将提高准确地将细胞外囊泡作为几种疾病的生物标志物来源的能力。目前用于隔离外来体的标准方法, 特别是离心和沉淀方法, 有几个缺点, 包括外切聚合和可溶性蛋白造粒 7, 17, 18。另外, SEC 的使用也显示了外切隔离的显著改善, 保护外来体不被聚合, 并改进了清除常见的污染物非 exos...

外来体是 nanovesicles (大小从30毫微米到150毫微米) 释放的几乎所有的细胞在人体内, 以促进细胞到细胞的通信进程 1, 2.有趣的是, 外来体的组成会根据原始单元格的变化以及单个3、4、5的健康状况而改变.此外, 外来体可以从几种生物液中提取, 如: 唾液、尿液和血液 2.由于这些特点, 外来体被认为是疾病生物标志物的良好来源。不幸的是, 没有标准的方法来隔离外来体。一些实验室考虑多级离心, 以 10万 x g 的最终高速步骤为外切隔离的金标准方法。然而, 最近的研究表明, 离心诱导外来体与可溶性蛋白和其他外来体的聚集, 除了影响外切完整性, 这两个可能会阻碍下游应用程序6, 7.外切隔离的其他常用方法包括但不限于: 以商业聚乙二醇 (PEG) 为基础的试剂、离心超滤和尺寸排除色谱法 (SEC) 沉淀。使用聚乙二醇 (PEG) 聚合物的商业试剂通过使外来体沉淀并形成颗粒而丰富了它们。使用这种聚合物的限制是残留的 PEG 聚合物和大量的可溶性非 exosomal 蛋白在最终产品的污染。超滤利用纤维素膜进行离心纯化浓缩囊泡;外来体保留在过滤器之上, 而较小的杂质和其他蛋白质通过膜 7, 8.就像其他方法一样, 离心超滤由于保留高水平的非 exosomal 蛋白, 包括蛋白质复合物和聚合体, 外来体的净化能力有限。最后, SEC 净化使用多孔树脂分离分子的大小。SEC 已经显示出有希望的结果, 通过捕获大多数的污染物蛋白和保持 exosomal 完整性, 克服了其他方法所遇到的大多数问题, 因为隔离是基于重力或低压系统7, 9。然而, 在 SEC 期间, 大蛋白质集料和脂蛋白的联合分离影响了最终外切制剂的纯度.虽然一些方法已经测试了外切纯化从细胞培养上清液和等离子7, 或仅等离子9, 11, 没有关于直接比较血液的方法的性能信息同一个人的血浆和血清。
在这里, 我们重点研究血液外来体的纯化, 通过比较各种工作流, 以确定是否囊泡浓缩技术是可翻译的血浆和血清。采用纳米微粒跟踪分析和印迹方法, 对最终产物中外切浓度、纯度和蛋白质组成的差异进行量化。最后的方法, 详细的在本协议中, 增加囊泡数和减少非 exosomal 蛋白水平。重要的是, 大量减少常见的共沉淀蛋白, 包括白蛋白和载脂蛋白。这两种蛋白质在血液中的高丰度以及它们与外来体共同纯化的频率导致了 "纯化" 样品之间的不一致, 从而扭曲了下游的分析结果。该协议包括使用商业上可用的 SEC 树脂;该树脂是由多孔珠组成, 其中蛋白质小于 700 kDa 可以进入珠。一旦内, 蛋白质由辛胺配体保留。洗脱液由外来体和大于700kDa 12 的分子组成。此外, 为了减少载脂蛋白的蛋白质聚集和抑制, 我们在工作流中加入了蛋白酶消化步骤。目前, 还没有单一的技术能够最大限度地提高外切产量, 同时减少共纯化非 exosomal 蛋白。本研究表明, 采用蛋白酶消化预处理与多种回收纯化相结合的纯化方法, 可提高血清和血浆外切的产量和纯度。

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Nanosight</td>
			<td style="width:149px;">Malvern</td>
			<td style="width:123px;">NS300</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Benchtop microcentrifuge&#160;</td>
			<td style="width:149px;">Thermo</td>
			<td style="width:123px;">Legend Mico21</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Benchtop centrifuge&#160;</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:123px;">Allegra 6R</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Waterbath&#160;</td>
			<td style="width:149px;">Benchmark</td>
			<td style="width:123px;">B2000-4</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Microplate reader</td>
			<td style="width:149px;">BIOTEK</td>
			<td style="width:123px;">Epoch</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">23227</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Micro BCA Protein Assay Kit</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">23235</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Phosphate buffered saline</td>
			<td style="width:149px;">Corning</td>
			<td style="width:123px;">21-040-CV</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">96 well plate</td>
			<td style="width:149px;">Corning</td>
			<td style="width:123px;">15705-066</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">UFC510096</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Amicon Ultra-4 Centrifugal Filter Units</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">UFC800324</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Capto Core 700</td>
			<td style="width:149px;">GE Healthcare</td>
			<td style="width:123px;">17548103</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Poly-Prep&#160;Chromatography Columns</td>
			<td style="width:149px;">BIORAD</td>
			<td style="width:123px;">7311550</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">NP0323BOX</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Nitrocellulose Membrane, Roll, 0.2 &#181;m</td>
			<td style="width:149px;">BIORAD</td>
			<td style="width:123px;">1620112</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Proteinase K,&#160;Tritirachium album</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">539480</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">SimplyBlue SafeStain</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">LC6065</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">SIGMAFAST&#160;BCIP/NBT</td>
			<td style="width:149px;">Millipore-SIGMA</td>
			<td style="width:123px;">B5655</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">4-Chloro-1-naphthol</td>
			<td style="width:149px;">Millipore-SIGMA</td>
			<td style="width:123px;">C6788</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">Anti-CD63 Antibody (rabbit anti-human) with goat anti-rabbit HRP secondary antibody</td>
			<td style="width:149px;">SYSTEM BIOSCIENCES</td>
			<td style="width:123px;">EXOAB-CD63A-1</td>
			<td style="width:124px;">Primary and secondary antibodies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:281px;">ALB Antibody (F-10)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-271605</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:281px;">apoB Antibody (C1.4)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-13538</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:281px;">apoA-I Antibody (B-10)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-376818</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
	</tbody>
</table>

protein digestion, ultrafiltration, and size exclusion chromatography to optimize the isolation of exosomes from human blood plasma and serum

外来体是一种从所有细胞类型中释放出来的 nanovesicle, 可以从任何体液中分离出来。外来体的内容, 包括蛋白质和 rna, 对它们所派生的细胞是独一无二的, 可以作为疾病的指标。一些常见的浓缩协议, 包括离心, 产外来体可溶性蛋白污染物。具体地说, 我们发现血液中最丰富的蛋白质经常与外来体共同纯化, 并可能混淆下游蛋白质组研究, 挫败低丰度生物标志物候选者的识别。另外的关注是由于非 exosomal 蛋白水平的不一致表示, 外切蛋白量化 irreproducibility。这里详述的协议是为了去除与外来体共纯化的非 exosomal 蛋白, 增加了外切纯化过程的严格。采用五例献血者配对血浆和血清对五种方法进行比较。用纳米微粒跟踪分析和微二辛可宁酸蛋白检测结果表明, 采用超滤和粒径排除色谱相结合的复合协议, 可获得最佳的囊泡浓缩和可溶性蛋白去除。西方印迹被用来验证预期丰富的血液蛋白, 包括白蛋白和载脂蛋白, 已耗尽。

以下数据是使用五健康献血者配对血浆和血清样本获得的。为了确定各处理步骤的效果, 对所提出的协议进行了五种变异, 并对外切恢复和非外切蛋白去除进行了比较。方法全身扫描仪最包括: 1) 秒 700 kDa + 超滤 3 kDa;2) 超滤 100 kDa;3) 蛋白酶 K + 超滤 100 kDa;4) SEC 700 kDa + 超滤 100 kDa, 5) 蛋白酶 K + 超滤 100 kDa + 秒 700 kDa + 超滤 3 kDa。
总...

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Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

在这里, 我们提出了一个协议, 以净化外来体从血浆和血清与减少共同纯化的非 exosomal 血液蛋白。优化的协议包括超滤、蛋白酶处理和尺寸排除色谱。增强纯化外来体的好处下游分析, 包括更准确的定量的囊泡和蛋白质组特征。

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Research

JoVE Journal

Biochemistry

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

在线体积排阻和离子交换色谱法SAXS光束线

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

科研

生物化学

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

人类外周血神经细胞分离通过评估Rab10磷酸化来询问帕金森氏症相关的LRRK2激酶途径

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

离子交换色谱 (IEX) 与多角度光散射 (ALS) 相结合, 用于蛋白质分离和表征

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

微量和外显子的纸基预集中和分离

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

Analysis and Specification of Starch Granule Size Distributions

淀粉颗粒大小分布的分析和规格

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

超中心对等离子脂蛋白的分离和分析

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

通过荧光体积排阻色谱快速评估膜蛋白质量

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...