Name: optimized protocol for the extraction of proteins from the human mitral valve
Uploaded: 2017-06-14T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve at JoVE.com

意大利卫生部支持这项研究（RC 2013-BIO 15）。我们非常感谢Barbara Micheli的优秀技术援助。

在该方案中，尽管正常的超声心动图参数，在多器官移植（冷缺血时间4-12小时，平均6±2小时）内，从器官移植排除器官移植的技术或功能原因收集人心脏。他们被送到米兰的心血管组织银行Monzino心脏中心（意大利米兰），为主动脉瓣和肺动脉瓣的银行服务。二尖瓣后叶不用于临床目的，因此在从供体亲属获得知情同意后，在主动脉瓣和肺动脉瓣隔离期间收集二尖瓣后叶。移植和研究的组织仅在家长同意后收集;在同意书上，只有在不适合人类临床使用（ 即微生物，功能和血清学问题）的情况下，他们授权（或不）使用心脏组织进行研究，遵循伦理委员会的指导方针蒙齐诺心脏中心。 二尖瓣制备<ol><li>器官移植后尽早收集人体二尖瓣（冷缺血时间4-12 h）。 </li><li>在洁净室内，从含有冷（4℃）溶液（ 即盐水溶液或平衡介质Eurocollins或威斯康星州）的运输袋中取出心脏。将其放入桶中并放置在生物安全柜（生物危害垂直空气流，A级，良好生产规范（GMP）分类）中，以进行阀门准备。 </li><li>将心脏放在柜内的无菌一次性悬垂物上。使用无菌的一次性手术刀，在距离顶点约4厘米处的左心室和右心室的水平上将心脏完全切开，与其主轴垂直。 </li><li>移动升主动脉和肺动脉显示左心房顶。 </li><li>用无菌高压灭菌镊子和捡拾，切割左耳廓上的左心房顶部，使二尖瓣可见，并允许确定大二尖瓣（前）和小二尖瓣（后）。 注意:前外侧和后内侧合缝定义前叶和后区的边界。 </li><li>使用无菌高压灭菌剪刀和非创伤镊子，解剖左心房和围绕整个二尖瓣周围的心室壁厚度。 </li><li>确定主动脉瓣主动脉瓣连续性。 注意:左心室包含整个二尖瓣和和弦。 </li><li>将二尖瓣二尖瓣分离自二尖瓣后叶小叶，沿着插入与心室切开后叶（连合）。 </li><li> 在盐水溶液中洗后叶。将小叶切成小块（&lt;1厘米2 ），并单独包裹在铝箔上。用液氮快速冻结。 注意:使用液氮时应遵循组织安全程序。 <ol><li>在程序结束时，用70％异丙醇溶液和6％过氧化氢溶液对柜子进行消毒。 </li></ol></li></ol>蛋白质提取<ol><li>使用镊子拿起储存在液氮中的样品，并立即将其放在干冰上，同时仍然包裹在铝箔中。在转运期间不要让样品解冻。 </li><li>在研磨之前，将研磨机系统（ 例如， CryoGrinder）的瓷/锆砂浆和样品与样品一起放入含有液氮（〜500 mL）的杜瓦瓶中。 注意:使用液氮时应遵循组织安全程序。 </li><li>将砂浆和杵放在含干冰的聚苯乙烯箱中。从铝箔上取出样品并放入砂浆中。 </li><li> 研磨他用大杵取样15-15次，用螺丝刀旋转杵。在研磨过程中将样品与预先冷却的铲刀的尖端混合。 <ol><li>用小杵重复。 </li></ol></li><li> 将研磨的样品转移到先前称重的管（ 例如 15mL离心管）上，通过倒置管，将其放置在研钵上，并将它们反转到一起以将样品移动到管中。使用预冷的铲子从砂浆中回收所有材料。 <ol><li>将样品保持在干冰上，以避免样品在转移过程中解冻。 </li></ol></li><li>计算样品的净重。 </li><li>在每个样品后清洁砂浆和杵，并通过高压灭菌或在200℃加热2小时对其进行去污。 </li><li>通过反转将粉末样品从离心管转移到均化器的玻璃管中。 </li><li>加入过滤的尿素缓冲液将r（8M尿素，2M硫脲，4％w / v CHAPS，20mM Tris和55mM二硫苏糖醇）加入到玻璃管中，每10mg粉状组织200μL尿素缓冲液。 注意:离心管中残留的粉末样品可以使用部分计算的尿素缓冲液体积进行回收。 </li><li>使用装有硼硅酸盐玻璃研钵和聚四氟乙烯（PTFE）杵的搅拌器均匀化样品。用搓动（1500转/分钟）将杵慢慢按压到样品上10次。 </li><li>回收上清液并将其转移到干净的1.7 mL离心管中。再次用新鲜的尿素缓冲液提取剩余的样品，加入第一次提取过程中使用的体积的一半。 </li><li>重复步骤2.10。 </li><li>回收上清液并将其与步骤2.11的上清液组合。将合并的上清液放在管旋转器上30分钟。 </li><li>在13,000 xg和4℃下离心管30分钟。 </li><li>恢复上清液d根据制造商的说明书使用Bradford蛋白测定法测量蛋白质浓度。将样品储存在-80°C直到使用。 </li></ol>

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作者没有什么可以披露的。

该方案的一个关键步骤是使用液氮冷冻样品并冷却研磨机系统。液氮的使用可防止生物降解并有效地进行粉末化，但需要对培训进行安全处理。 在该协议中，具有用于样品研磨的研磨机系统，因为小样品难以从标准的砂浆和杵中回收。在这种情况下，小样品作为细粉扩散到砂浆表面，使收集困难。另一个优点是研磨机是电动的，这允许以可再现的方式处理更多数量的样品并?...

最近的证据改变了对mRNA合成后发生的许多调节机制的作用的理解。实际上，翻译，转录后和蛋白水解过程可以调节蛋白丰度和功能。假设转录本水平是蛋白质丰度的主要决定因素，mRNA的浓度代表相应蛋白质的代谢 - 已被部分修订。目前，转录本水平仅部分预测蛋白质丰度，这表明转录后事件发生调节细胞内的蛋白质1,2 。 此外，蛋白质最终决定细胞的功能，因此决定其表型，其可以经受自分泌，旁分泌和内分泌因子的响应的动态变化;血液介体;温度;药物治疗和疾病发展换货。因此，以蛋白质水平为重点的表达分析有助于表征蛋白质组，并且揭示其作为疾病发病机理的一部分发生的关键变化3 。 因此，尽管现有的技术挑战，蛋白质组学所提出的澄清健康和疾病状况的机会也是艰巨的。蛋白质组学可以贡献的特别有希望的研究领域包括:在任何水平（ 即全细胞或组织，亚细胞区室和生物液体）上鉴定改变的蛋白质表达;用于疾病诊断和预后的新型生物标志物的鉴定，验证和验证;并希望能够鉴定可用于治疗目的的新蛋白质靶标，以及药物功效和毒性的评估。 捕捉复杂性蛋白质组是技术挑战。目前的蛋白质组学工具提供了进行大规模，高通量分析来鉴定，定量和验证改变的蛋白质水平的机会。此外，引入分馏和富集技术，旨在避免由最丰富的蛋白质引起的干扰，还通过包括最少丰富的蛋白质来改善蛋白质鉴定。最后，蛋白质组学被补充了翻译后修饰的分析，其逐渐成为蛋白质功能的重要调节剂。 然而，分析中的生物标本中的样品制备和蛋白质回收仍然是蛋白质组学工作流程中的限制步骤，并增加可能陷阱的潜力5 。事实上，在大多数必须优化的分子生物学技术中，第一步是组织匀浆离子和细胞裂解，特别是在不存在扩增方法的低丰度蛋白质的分析过程中。此外，蛋白质的化学性质可以影响其自身的恢复。例如，高疏水性蛋白质的分析是非常具有挑战性的，因为它们在等电聚焦过程中容易沉淀，而跨膜蛋白几乎不溶（参见参考文献5）。此外，组织组成变异性为开发通用提取方法创造了显着的障碍。最后，由于几乎所有临床标本的数量都是有限的，所以必须以最小的样品量使蛋白质制备具有最大的恢复和重现性。 这项工作描述了从正常人心脏二尖瓣进行蛋白质提取的优化方案，这代表了蛋白质组学分析的非常具有挑战性的样品。正常二尖瓣是一种lex结构位于左心房和心脏左心室之间（ 图1 ）。它在控制从心房到脑室的血液流量方面起着重要的作用，防止回流，并确保适当的氧气供应给全身，从而保持足够的心输出量。然而，它通常被认为是一种"无活性"组织，细胞率低，组成少，主要是细胞外基质。这是因为在正常情况下，驻留的瓣膜间质细胞（VIC）具有低蛋白质生物合成速率7的静止表型。 然而，已经证明，在病理状态下，海绵体中的VIC的数量增加并且其蛋白质合成被激活，以及其他功能和表型变化8 。因此，最少的数据可用并不奇怪文献集中于病理性二尖瓣9,10的分析，其中活化的VIC的增加的数量可能解释相对较高数量的鉴定的蛋白质。 总之，本议定书可能通过研究二尖瓣蛋白组分来发展对负责二尖瓣疾病的致病机制的理解。事实上，更深入地了解潜在的病理过程可能有助于改善瓣膜疾病的临床管理，其目前的干预指标主要取决于血液动力学考虑因素。

<table><tbody><tr><td>Saline solution</td><td></td><td></td><td>0.9 % NaCl</td></tr><tr><td>Eurocollins A</td><td>SALF</td><td>30874046</td><td>Balanced organ&#039;s transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins</td></tr><tr><td>Eurocollins B</td><td>SALF</td><td>30874022</td><td>Balanced organ&#039;s transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins</td></tr><tr><td>Wisconsin</td><td>Bridge life</td><td>RM/N 4081</td><td>Balanced organ&#039;s transport medium</td></tr><tr><td>Biohazard vertical flow air</td><td>Burdinola</td><td></td><td>Class A GMP classification</td></tr><tr><td>Dewar Flask</td><td>Thermo Scientific</td><td>Nalgene 4150-1000</td><td></td></tr><tr><td>Cryogrinder system</td><td>OPS diagnostics</td><td>CG 08-01</td><td>Grinder system containing mortars, pestles and screwdriver</td></tr><tr><td>Stainless steel forceps</td><td></td><td></td><td></td></tr><tr><td>Stainless steel spatula</td><td></td><td></td><td></td></tr><tr><td>Disposable sterile scalpel</td><td>Medisafe</td><td>MS-10</td><td></td></tr><tr><td>Stainless steel scissors</td><td></td><td></td><td>Autoclavable</td></tr><tr><td>Stainless steel picks</td><td></td><td></td><td>Autoclavable</td></tr><tr><td>Disposable sterile drap</td><td>Mon&amp;amp;Tex</td><td>3.307.08</td><td></td></tr><tr><td>Sterilizing solution with isopropyl alcohol</td><td></td><td></td><td>70% isopropyl alcohol</td></tr><tr><td>Sterilizing solution with hydrogen peroxide</td><td></td><td></td><td>6% hydrogen peroxide</td></tr><tr><td>Micropipette, 1 mL, with tips</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>VWR international</td><td>9278</td><td></td></tr><tr><td>1.7 mL centrifuge tubes</td><td>VWR international</td><td>PIER90410</td><td></td></tr><tr><td>Urea buffer</td><td></td><td></td><td>8 M urea, 2 M thiourea, 4 % w/v CHAPS, 20 mM Trizma, 55 mM Dithiotreitol</td></tr><tr><td>Urea</td><td>Sigma aldrich</td><td>U6504-1KG</td><td>To be used for Urea buffer</td></tr><tr><td>Thiourea</td><td>Sigma aldrich</td><td>T8656</td><td>To be used for Urea buffer</td></tr><tr><td>CHAPS</td><td>Sigma aldrich</td><td>C3023-5GR</td><td>To be used for Urea buffer</td></tr><tr><td>Dithiotreitol</td><td>Sigma aldrich</td><td>D0632-5G</td><td>To be used for Urea buffer</td></tr><tr><td>Syringe 50 mL</td><td>PIC</td><td></td><td>To be used to filter Urea buffer</td></tr><tr><td>0.22 &amp;micro;m filter</td><td>Millipore</td><td>SLGP033RB</td><td>To be used to filter Urea buffer</td></tr><tr><td>PFTE Pestle, 2 mL</td><td>Kartell</td><td>6302</td><td>Part of Potter-Elvehjem homogenizer</td></tr><tr><td>Borosilicate glass mortar</td><td>Kartell</td><td>6102</td><td>Part of Potter-Elvehjem homogenizer</td></tr><tr><td>Stirrer</td><td>VELP scientifica</td><td>Stirrer DLH</td><td>To be used for homogenization by Potter-Elvehjem</td></tr><tr><td>Bradford Protein assay</td><td>Bio-Rad laboratories</td><td>5000006</td><td></td></tr><tr><td>Tube rotator</td><td>Pbi International</td><td>F205</td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Polystyrene box</td><td></td><td></td><td></td></tr><tr><td>Dry ice</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td>For centrifugation of 1.7 mL centrifuge tubes at 13,000 x g</td></tr><tr><td>Freezer -80&amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>Precision balance</td><td></td><td></td><td></td></tr><tr><td>Autoclave</td><td></td><td></td><td>For sterilization</td></tr><tr><td>Cryogenic gloves for liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Professional forced ventilation and natural air convection oven</td><td></td><td></td><td>For sterilization</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma aldrich</td><td>P8340-5ML</td><td>100X solution</td></tr><tr><td>ProteoExtract Protein Precipitation Kit</td><td>Calbiochem</td><td>539180</td><td></td></tr><tr><td>RapiGest</td><td>Waters</td><td>186001861</td><td></td></tr><tr><td>Cytoscape</td><td>www.cytoscape.org</td><td>version 2.7</td><td>Software platform for Gene Ontology analysis</td></tr><tr><td>BiNGO</td><td>http://apps.cytoscape.org/apps/bingo</td><td>version 3.0.3</td><td>Plugin for Gene ontology analysis</td></tr><tr><td>AlphaB Crystallin/CRYAB Antibody</td><td>Novus Biologicals</td><td>NBP1-97494</td><td>Mouse monoclonal antibody against CryAB</td></tr><tr><td>Septin-11 Antibody</td><td>Novus Biologicals</td><td>NBP1-83824</td><td>Rabbit polyclonal antibody against septin-11</td></tr><tr><td>FHL1 Antibody</td><td>Novus Biologicals</td><td>NBP-188745</td><td>Rabbit polyclonal antibody against FHL-1</td></tr><tr><td>Dermatopontin Antibody</td><td>Novus Biologicals</td><td>NB110-68135</td><td>Rabbit polyclonal antibody against dermatopontin</td></tr><tr><td>Goat Anti mouse IgG HRP</td><td>Sigma aldrich</td><td>A4416-0.5ML</td><td>Secondary antibody for immunoblotting</td></tr><tr><td>Goat Anti rabbit IgG HRP</td><td>Bio-Rad laboratories</td><td>170-5046</td><td>Secondary antibody for immunoblotting</td></tr></tbody></table>

optimized protocol for the extraction of proteins from the human mitral valve

细胞蛋白质组学的分析可以帮助阐明基于疾病的分子机制，这是由于技术的发展，允许大规模鉴定和定量复制生物系统中存在的蛋白质。从蛋白质组学方法获得的知识有可能导致更好地了解基础疾病的致病机制，允许鉴定新的诊断和预后疾病标志物，以及希望的治疗靶点。然而，心脏二尖瓣代表蛋白质组学分析的非常具有挑战性的样品，因为蛋白多糖和富含胶原蛋白的细胞外基质的细胞数量较少。这使得挑选提取蛋白质进行全球蛋白质组学分析。该工作描述了与随后的蛋白质分析相容的方案，如定量蛋白质组学和免疫印迹。这可以允许数据关联的相关性g蛋白表达与定量mRNA表达和非定量免疫组化分析数据。实际上，这些方法在一起执行时，可以更全面地了解基因疾病的基因分子机制，从mRNA到翻译后蛋白质的修饰。因此，这种方法可以与对心脏瓣膜病理学研究感兴趣的研究者有关。

蛋白质在尿素缓冲液中的提取和溶解与基于等电聚焦（二维电泳（2-DE） 11和液相等电聚焦（IEF） 12 ）的蛋白质组学方法直接相容，并且在Laemmli缓冲液13中稀释后进行免疫印迹含有蛋白酶抑制剂混合物14 。 对于基于无凝胶质谱法（ 即与数据无关质?...

Watch this Scientific Journal Video about Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve at JoVE.com

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

人二尖瓣的蛋白质组成仍然是部分未知的，因为其分析由于细胞数量低，因此通过低蛋白质生物合成而复杂化。这项工作提供了有效提取蛋白质用于二尖瓣蛋白质组分析的方案。

从人体二尖瓣提取蛋白质的优化方案

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

optimized-protocol-for-extraction-proteins-from-human-mitral

Research

JoVE Journal

Biochemistry

12.8K 次观看.  Centro Cardiologico Monzino IRCCS. 人二尖瓣的蛋白质组成仍然是部分未知的，因为其分析由于细胞数量低，因此通过低蛋白质生物合成而复杂化。这项工作提供了有效提取蛋白质用于二尖瓣蛋白质组分析的方案。 

视频: Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK 细胞 Mitoribosome 的快速分离

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

骨骼肌线粒体蛋白质导入能力的测定

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

<meta charset="utf8"></head><body>从啮齿动物中分离线粒体以进行高质量的生物能量研究

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>作者聚焦:揭示线粒体磷酸钙在心力衰竭和生物能量学中的作用

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

从啮齿动物心脏组织中稳健地分离线粒体 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

生物学

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

从人体二尖瓣提取蛋白质的优化方案

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一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定

从发展胚胎小鼠心脏瓣膜区蛋白分离