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注意:此方法以前报告10 。 <ol><li>准备新鲜的分离缓冲液（IB），pH 7.4; 2M蔗糖; 1M蔗糖/ 0.1mM CaCl 2 ;和0.1mM CaCl 2 （ 参见表1 ）。在冰上冷却解决方案。 </li><li>萨通过颈椎脱位引起五只小鼠。斩首并迅速清除每只动物的大脑。解剖感兴趣的脑区域（即，海马，0.25-0.35g新鲜组织），并将其置于5mL Potter-Elvehjem玻璃管中，冰上加入1mL IB。 </li><li>使用均质搅拌器（10冲程，以700-900转/分钟）均质化组织，优选在冰浴中排除样品加温。 </li><li>将匀浆的组织（1mL）放入含有6mL 2M蔗糖和2.5mL 0.1mM CaCl 2的 15mL管中，4℃。通过翻转管慢慢混合。 注意:钙的添加对于维持细胞器之间的粘合剂相互作用是至关重要的，因此保持不同"密度"的结构。 </li><li>将溶液放入12 mL离心管中，缓慢加入2.5 mL 1 M蔗糖/ 0.1 mM CaCl 2置于各管顶部，形成蔗糖梯度。 注意:标记位置of使用永久标记在离心管上的梯度界面。 </li><li>称量和平衡离心管与IB溶液在他们相应的钢支架和他们的闭合盖。 </li><li>使用摆动式转子离心机将管子在100,000xg和4℃下离心3小时。用所有的钢支架完全充满转子（如果需要，甚至是空的）。 </li><li>丢弃含有髓磷脂的顶层。使用巴斯德吸管，收集对应于突触体部分的1.25-M和1-M蔗糖间期的白色环。 </li><li>使用IB在离心管中稀释9倍体积的突触体。 </li><li>称量和平衡离心管与IB溶液在他们相应的钢支架和他们的闭合盖。 </li><li>使用摆动转子离心机将管子以15,000xg和4℃离心30分钟。 </li><li>丢弃上清液并重悬用1.1mL IB溶液沉淀。收集100μL的这种突触体溶液，并在11,000 x g和4ºC下离心5分钟。 </li><li>将突触体沉淀重悬于5％十二烷基硫酸钠（SDS）中，并将该样品储存在-20℃。 注意:该样品将对应于进一步的Western印迹分析的总突触体分数。剩余的突触体部分（〜1 mL）可以在-20°C下冷冻直到使用或进行后续的亚突触分选。突触体或纯化的突触占总海马体积的1％和2％。 </li></ol>前，后和外突的隔离<ol><li>准备2x新鲜增溶缓冲液，pH 6.0; 1x溶解缓冲液，pH 6.0;溶解缓冲液，pH 8.0;和0.1mM CaCl 2 （ 参见表1 ）。在冰上冷却解决方案。 注意:这些溶液的pH应该是准确的y调整以实现良好的亚突触分选。 </li><li>用5 mL 0.1 mM CaCl 2缓慢稀释1 mL重悬注射突变体部分（见步骤1.12）。 </li><li>加入相同体积（5 mL）冰冷的2x溶解缓冲液（pH 6.0），并在冰上在高搅拌下，在冰上的烧杯中孵育50分钟。 注意:虽然1％的Triton X-100在pH6.0溶解了所有的质膜蛋白质，它保留了突触接触点或结点内的蛋白质（ 即，突触前和突触后结构）。 </li><li>将溶液置于离心管中。称重并平衡管中的等量体积的0.1mM CaCl 2和2x溶解缓冲溶液，pH 6.0，在其相应的钢支架内及其闭合盖。 </li><li>使用摆动式转子离心机将管子以40,000xg和4℃离心30分钟。所得上清液代表突触后部分，并且颗粒对应于突触结（ 即，突触前和突触后部分）。 </li><li>将含有超弹性组分的上清液浓缩至最终200μL体积，使用在4,000xg和4℃离心的15mL 10K过滤管。 </li><li>将所得的浓缩超弹性组分（200μL）在-20℃下沉淀5次（1mL）预冷（-20℃）丙酮过夜。 </li><li>第二天，以18,000 x g和-15°C离心超刺激级分30分钟。离心后，弃去丙酮上清液，干燥颗粒以除去任何痕量的丙酮。 </li><li>最后，用200μL的5％SDS重新悬浮含有突触前蛋白质的沉淀。超声处理颗粒，如果需要。 </li><li>在不破坏它的情况下，用2 mL 1x溶解缓冲液（pH 6.0）仔细清洗含有突触前和突触后部分的沉淀。丢弃缓冲区。 </li><li>重悬使用玻璃巴斯德吸管，用10mL的1x溶解缓冲液（pH8.0）沉淀。 注意:尽管1％Triton X-100 pH 8.0可溶解突触前专业化，但仍保留不溶性突触后密度4 。 </li><li>在高搅拌下，在冰上的烧杯中，将悬浮液在冰上孵育50分钟。 </li><li>将溶液置于离心管中。称重并平衡管子与1x溶解缓冲溶液，pH 8.0，在它们相应的钢支架内和与他们的封闭盖子。 </li><li>使用摆动转子离心机将管子以40,000xg和4℃离心30分钟;所得到的上清液对应于突触前部分和沉淀到突触后部分。 </li><li>处理含有突触前部分的上清液，如步骤2.6-2.9所述。 </li><li>用200μL的5％SDS重悬含有突触后部分的沉淀。超声处理颗粒，我f需要。 </li></ol> 3.通过免疫印迹分析样品以验证膜分级<ol><li>使用二金鸡宁酸蛋白测定法确定每个级分中的蛋白质的量（ 即，总，外，前和突触后部分）。 </li><li>从SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）样品缓冲液中取每个级分的20μg蛋白质并稀释至最终体积为50μL。在100ºC煮沸5分钟。 </li><li>通过SDS-PAGE电泳分离蛋白质10％，在还原条件下用4％浓缩凝胶分离。在恒定电压80 V下电泳，直到染料进入下凝胶，然后将电压提高到120 V. </li><li>将蛋白质转移到PVDF膜上，并在室温下连续摇动，用IB封闭溶液封闭45分钟。 </li><li>在4℃下用指定的一抗（ 即抗SNAP-25，抗PSD-95，抗突触泡蛋白或抗GPR37）在IB封闭溶液中连续振荡稀释。 </li><li>用IB洗涤溶液洗涤膜三次（每次10分钟），以消除未结合的一抗。 </li><li>在暗条件下，在室温下连续振荡，与指定的二抗孵育90分钟。 </li><li>用IB洗涤溶液洗涤膜三次（每次10分钟）以消除未结合的二抗。 </li><li>用化学发光底物孵育膜（在黑暗条件下制备混合物，并按制造商提供的溶液A和B的1:1比例）。 </li><li>分析化学发光检测装置中的膜。 </li></ol>

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作者没有什么可以披露的。

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）来进行成功的分级;否则，产量太低，不能评估正在研究的蛋白质的身份以及定位。 相反，如果使用过量的脑组织，则分离程序w生病不是最佳的。仔细调整溶液的pH以确保最佳的亚突触分馏也是至关重要的。因此，每次进行脑膜分离时，必须在任何进一步研究之前验证每个部分的效率。重要的是，以下方案由于其差异结构和分布而不太可能适用于抑制性突触的亚突触分选。然而，所有这些缺点并不能掩盖这个实验程序的巨大用处，毫无疑问，这一过程将成为研究突触的通用方法。

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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			<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>
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		<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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		<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>
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			<td height="21" style="height:21px;width:128px;">Non fat dry milk</td>
			<td style="width:249px;"></td>
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			<td style="width:167px;"></td>
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			<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>
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		<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>
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		<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>
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			<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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			<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>
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		<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:128px;">Sonifier 250</td>
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			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
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			<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>
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		<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>
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			<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>
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		<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>
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			<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>
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			<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>
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		<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肽。在其位置，GPR37被提议由头活化剂p激活顺铂14，15，16，最近由神经肽类proaposin和prosaptide 17 ，虽然这些关联尚未被普遍接受。 GPR37与帕金森病的联系受到最多关注18 。因此，在正常和病理状况下，都有意识地了解这种有趣的受体的神经生物学。因此，揭示GPR37亚突触定位可能有助于阐明其在脑内的功能。为此，首先在8周龄时从C57BL / 6J（WT）和GPR37-KO小鼠中分离出海马。然后，使用膜分离方案（参见图1的程序的示意图）纯化亚突变前后部分。随后，这些亚突触室的纯度由各突触标记的分离: i ）突触前水泡标记（突触泡蛋白）; ii ）突触前活动区标记（SNAP-25）;和iii ）突触后密度标记（PSD95）。因此，通过使用针对这些蛋白质的特异性抗体的免疫印迹分析突触前蛋白，SNAP-25和PSD95在亚突变前后分泌物中的富集（ 图2 ）。因此，对于所测试的每个突触标记，发现至少90％的分馏效率（ 图3 ），类似于前述6 。有趣的是，与突触前（20±4％）和突触后（36±2％）级分（ 图3 ）相比，外淋巴组分中GPR37的免疫反应性更丰富（n = 3; P &lt;0.001）。另外，我们的数据表明，虽然存在于突触前活动区，GPR37主要是lo在突触后密度（n = 3; P &lt;0.05）（ 图3 ）。总体而言，脑膜分离方案允许评估小鼠海马中GPR37的亚突触分布，从而为未来对这种孤儿受体的操作提供有价值的信息。 <img alt="图1" src="/files/ftp_upload/55661/55661fig1.jpg" /> 图1:膜分馏方案的示意图流程图。所有实验步骤都在左侧列中进行描述，而样品集合在右侧列中描述。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55661/55661fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55661/55661fig2.jpg" /> 数字2:GPR37在小鼠海马中的突触后分布。代表性的免疫印迹显示Synaptophysin，SNAP-25和PSD-95作为额外的，突触前和突触后特异性突触标记，以及WT和GPR37-KO小鼠的海马突触部分中的GPR37免疫反应性。海马突触体（Syn）被分级为突触前（Extra）和突触前（Pre）活动区和突触后密度（Post）分数。使用兔抗突触泡蛋白（1:3,000），小鼠抗SNAP-25（1:3,000），兔抗PSD95（1:3,000）和兔抗 - 突触泡蛋白（1:3000），通过免疫印迹（20μg蛋白质/泳道） -GPR37（1μg/ mL）抗体。使用HRP缀合的山羊抗兔（1 / 30,000）或兔抗小鼠（1:30000）抗体检测初级结合的抗体。这些数据在许可下从参考文献10中提取。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55661/55661fig2large.jpg" target="_blank">请点击h</a>以查看这个数字的较大版本。 <img alt="图3" src="/files/ftp_upload/55661/55661fig3.jpg" /> 图3:相对定量的GPR37富集海马额外，前和突触后部分。通过光密度扫描测量对应于图2所示的超突触（额外，黄色柱），突触前（前，绿色柱）和突触后（柱后红柱）级分的免疫印迹膜上的免疫反应谱带的强度。密度从非饱和条带量化。使用在最富集部分中的突触泡蛋白，SNAP-25，PSD95和GPR37的量将数值归一化（以相对密度计扫描的RDS的百分比），并作为三次独立实验的平均值±SEM表示。星号表示显着不同的数据:* p &lt;0.05，*** p &lt;0。001（带有Bonferroni 事后检测的单因素方差分析）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55661/55661fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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 ...

brain-membrane-fractionation-an-ex-vivo-approach-to-assess

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

10.3K 次观看.  Universitat de Barcelona, L'Hospitalet de Llobregat. 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。 

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

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

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

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

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

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

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

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

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 ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

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

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and 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

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

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

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

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 ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

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

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

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

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient 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

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

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 ...