Name: brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization
Uploaded: 2017-05-12T15:00:00
Description: Watch this Scientific Journal Video about Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization at JoVE.com

这项工作得到了经济部长萨洛德·卡洛斯三世（SAF2014-55700-P，PCIN-2013-019-C03-03和PIE14 / 00034）的支持，加拿大皇家科学院院士（ICREA Academia-2010） ）和代理商Innovatie门Wetenschap en Technologie（SBO-140028）到FC另外，X. M，VF-D。和FC属于“神经药理学和疼痛”认可研究组（Generalitat de Catalunya，2014 SGR 1251） 。这项工作也得到了CAPES（巴西）对FC的“Programa Pesquisador Visitante Especial-Ciênciasem Fronteiras”的支持

所有动物实验程序均由巴塞罗那动物使用和护理委员会（CEEA）批准，符合“实验动物护理和使用指南” 11和遵循欧洲共同体第86/609 / CCE，FELASA和ARRIVE指南。因此，将小鼠容纳在标准的笼子中， 随意获得食物和水，并保持在受控的标准条件下（从7:30 AM，22℃温度和66％湿度开始12小时黑暗/轻循环）。 1.使用不连续的蔗糖梯度获取小鼠脑突触体<p class="j...

<ol>
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这里提出的方案构成了一个强大的生物化学工具，用于研究任何脑区域内特异性蛋白质亚突触分布。然而，在技术上固有的一些缺点在此值得强调。例如，主要的限制之一是净化合理量的蛋白质所需的相对较大量的组织以便对所有亚突触部分进行免疫印迹分析。这个问题可能与突触（ 即突触体）仅占总海马体积的1-2％的事实有关。实际上，需要1到1.5g的新鲜组织（ 即海马体）来进行...

突触传播取决于突触的身体完整性，早在1897年由福斯特和谢林顿1设想的概念。因此，了解关键的神经传递组分（ 例如离子通道，受体等 ）的分布对于在正常和病理状况下阐明突触功能至关重要。电子显微镜（EM）对原型中枢神经系统（CNS）突触的当前超微结构概念作出了巨大贡献。以这种方式，EM已经精细地建立了突触前和突触后密度之间的差异，这些差异被相当均匀的距离（〜25nm） 2的裂缝隔开。有趣的是，突触后装置在其质膜下方表现出相对连续的电子致密增厚，即所谓的突触后密度或PSD2。相反，在突触前的装置，一个警告e不连续的细胞质网络布置在质膜正下方，这对于突触囊泡与质膜活性区3的对准和对接是必不可少的。因此，EM构成调查结构保守的CNS突触中蛋白质分布的黄金实验方法。然而，由电子显微照片提供的信息是静态的。实际上，积累的证据表明， 体内突触是非常动态的，因此在持续突触传播时经历剧烈的结构变化。此外，突触的形态和组成可以在不同的CNS区域以及发育，成熟，老化和神经病理学病症的发展中发生变化。总的来说，一项专注于分离属于不同突触室的蛋白质的生物条件的方案代表了一种更全面的突触功能研究的宝贵工具。 等人 （2001） 4 ，基于pH偏移，其削弱在突触前和突触后装置中发生的粘合相互作用。首先，通过在pH6.0下使用温和的洗涤剂，可以辨别保持突触前和突触后装置的粘附连接，并且可以从突触外膜结构域维持，其可以被溶解并因此可以从突触接触中提取。随后，在温和的洗涤剂存在下，将pH从6.0升高至8.0减弱了将突触前活性区域紧密结合到突触后密度的粘附连接点的强度。因此，突触前隔室是如此活化并且可以与突触后密度分离，这主要是由于所用洗涤剂的浓度不能促进其溶解而保留4 。有趣的是，最终高于90％的分离效率可以通过不同的亚突触标记来证实： i ）突触前活动区的突触体相关蛋白25（SNAP-25）; ii ）来自突触后部分的突触素（ 即活性区外，包括微粒体）;和iii ）突触后密度蛋白95（PSD-95）。值得注意的是，这种脑膜分离方法已被成功应用。因此，可以精确地确定不同受体的亚突触定位，例如α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体5 ，腺苷A 1受体（A 1 R） 6 ，腺苷A 2A受体（A 2A R） 7 ，三磷酸腺苷（ATP）P2受体8 ，烟碱乙酰胆碱受体亚基9和帕金森病相关受体GPR37 10 。然而，许多限制可能阻碍对特定神经元蛋白的突触分布的适当评估。因此，在这个过程中，我们不仅完整地描述了整个方案，而且还强调了要考虑的一些关键点，例如需要相当大量的组织，低蛋白质产量以及验证效率的强制性要求在进行确定的实验之前进行每次分离。

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		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sucrose</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,211,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">CaCl2</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">2,112,211,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">MgCl2&#183;6H2O</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,313,961,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Protease inhibitor cocktail Set III</td>
			<td style="width:249px;">Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">535140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Trizma Base</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tris-HCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,236,541,209</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Triton X-100</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">X100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">SDS</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">L3771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Glycerol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">G5516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Bromophenol Blue</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,311,651,604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Dithiothreitol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">D0632</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tween 20</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">P2287</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Non fat dry milk</td>
			<td style="width:249px;"></td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:128px;">NaCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,216,591,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,314,941,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KH2PO4</td>
			<td style="width:249px;">Merck</td>
			<td style="width:160px;">4873</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Na2HPO4</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,781,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Basic 20 pH</td>
			<td style="width:249px;">Crison, Alella, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Polytron VDI 12 Adaptable Homogenizer</td>
			<td style="width:249px;">VWR, Radnor, PA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
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		<tr height="65">
			<td height="65" style="height:65px;width:128px;">Ultra-Clear Tubes (14x89mm)</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;">344059</td>
			<td style="width:167px;">Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Amicon Ultra-15 Centrifugal filters Ultracel -10K</td>
			<td style="width:249px;">Merck Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">UFC901008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Centrifuge 5430R</td>
			<td style="width:249px;">Eppendorf, Hamburgo, Germany</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Optima L-90K Ultracentrifuge</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sonifier 250</td>
			<td style="width:249px;">Branson, Danbury, Connecticut</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Amersham Imager 600</td>
			<td style="width:249px;">GE Healthcare Europe GmbH, Barcelona, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Disposable Glass Pasteur Pippetes 230 mm</td>
			<td style="width:249px;">VWR, Radnor, PA, USA</td>
			<td style="width:160px;">612-1702</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Compact Balance EK-610</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Pierce&#8482; BCA Protein Assay Kit</td>
			<td style="width:249px;">Pierce Biotechnology, Rockford, IL, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">SuperSignal west pico chemiluminescent substrate</td>
			<td style="width:249px;">Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">GR 200 Precision Balance</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-GPR37</td>
			<td style="width:249px;">Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies used at a final concentration of 0.250ug/ml</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-SNAP-25, anti-PSD-95, anti-synaptophysin</td>
			<td style="width:249px;">Abcam, Cambridge, United Kingdom</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-mouse IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-rabbit IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:30000</td>
		</tr>
	</tbody>
</table>

brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization

评估突触蛋白的组成和功能是神经科学中的重要挑战。然而，评估突触内发生的神经传递是不容易的，因为它被动态蛋白质 - 蛋白质相互作用和磷酸化事件高度调控。因此，当使用任何方法研究突触传播时，主要目标是保留这些瞬时生理修饰。在这里，我们提出一个脑膜分离方案，代表一个强大的程序来分离属于不同突触室的蛋白质。换句话说，该方案描述了从突触前，突触后和突触后室进行蛋白质富集的生物化学方法。首先，突触体或突触末端通过不连续的蔗糖梯度从包含所有突触室的神经元获得。值得注意的是，这种初始突触膜制备的质量是critical。随后，通过在不同pH条件下使用温和的去污剂进行轻溶解来实现不同亚突触室的分离。这允许通过梯度和等离子体离心分离。最后，通过使用良好表征的突触蛋白标记（ 即， SNAP-25，PSD-95和突触素蛋白）的免疫印迹分析验证了不同亚突触间隔区（ 即，前，后和再突触后膜部分）的蛋白质富集，分别），从而能够直接评估任何特定神经元蛋白的突触分布。

所描述的方法主要用于神经元蛋白的亚突触分析，特别是用于突触受体5,6,7,8,9的分离和生物化学表征。有趣的是，这里显示的代表性结果显示了该实验程序对孤立G蛋白偶联受体的亚突触海马分布的分析的有用性，即帕金森病相关受体GPR37 10 。 GPR37最初是通过搜索内皮素和铃蟾肽受体13的同系物来鉴定的，尽管发现它不结合内皮素或相关?...

Watch this Scientific Journal Video about Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization at JoVE.com

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

在这里，我们提出一个脑膜分离方案，代表一个强大的程序来分离属于不同突触室的蛋白质。

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Research

JoVE Journal

Biochemistry

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

大肠癌细胞中磷酸化和微管结合与 Tau 蛋白定位的测定

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

拟南芥叶绿体子隔层的制备及免疫印迹蛋白质组化分析

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

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 ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Polysome Profiling without Gradient Makers or Fractionation Systems

无需梯度测定器或分馏系统的多聚体分析

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

萌芽酵母酿酒酵母中软骨蛋白定位的评估

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...