这项工作得到了 Ministerio de Ciencia e Innovación （https://ciencia.sede.gob.es/） 的资助号“PGC2018-095795-B-I00”和阿拉贡总局 （DGA） （https://www.aragon.es/） 对 PF-S 和 RM-L 的资助号“Grupo de Referencia： E35_17R”和资助号“LMP220_21”。

注： 表 1 中指定了所有培养基和缓冲液的成分，与本协议中使用的所有材料和试剂相关的详细信息列在 材料表中。
1. 从细胞培养物中分离线粒体
注意：检测的细胞的最小体积为 ~30-50 μL 的浓缩细胞（步骤 1.4）。这大约相当于至少两个或三个 100 mm 细胞培养板或一个汇合度为 80-90% 的 150 mm 板，具体取决于细胞类型（L929 来源细胞或 MDA-MB-468 中的 5 × 106 和 107 个细胞之间）。有效的细胞破损是一个关键步骤。
<ol><li>将贴壁细胞培养至约 80% 汇合，或将悬浮细胞培养至足够的密度。</li>
	<li>通过以 500 × g 离心 5 分钟收获细胞（在非贴壁细胞的情况下直接从细胞悬液中收获，或者在贴壁细胞的情况下，使用含 0.05% 胰蛋白酶和 0.02% EDTA 的胰蛋白酶-EDTA 溶液在 1x PBS 中直接从细胞悬液中收获细胞）。</li>
	<li>用冷的 1x PBS 洗涤细胞 2 次，并以 500 × g 离心 5 分钟沉淀。 注意：从这一点开始，所有步骤都必须在 4 °C 下进行。 因此，所有试剂都必须是冷的，并且含有细胞或线粒体的试管必须保存在冰上。</li>
	<li>第二次离心后，弃去上清液，估计细胞沉淀体积 （cpv），并将细胞在 -80 °C 下冷冻至少 15 分钟，以促进步骤 1.8 中的膜破裂。 注意：此时可以停止该方案，并且细胞可以在-80°C下储存数周。通常，10 或 15 mL 锥形底刻度管是合适的。</li>
	<li>让细胞在冰上缓慢解冻。</li>
	<li>将填充的细胞沉淀重悬于体积等于 cpv 7 倍的低渗缓冲液（10 mM MOPS，83 mM 蔗糖，pH 7.2）中（例如，在 700 μL 缓冲液中加入 100 μL 的填充细胞）。 注意：音量必须足以获得有效的 爆音（请参阅步骤 1.8 中的注释）。</li>
	<li>将细胞悬液转移到适当大小的 Potter-Dounce 匀浆器中，并在冰中孵育 2 分钟，让细胞膨胀。例如，对于 700-800 μL 的细胞悬液，1 mL 容量的均质器就足够了。</li>
	<li>通过在均质器中执行 8 到 10 次冲程来打破细胞膜，该匀浆器与以 600 rpm 旋转的电机驱动特氟龙杵相连。 注意：在进行中风时，重要的是要产生一个真空，这将提高细胞破裂效率以及分别用于溶胀和脆弱膜的低渗和冷冻作用。如果做得好，每次冲程快速拉下均质器时，会听到“砰”的一声。</li>
	<li>向细胞悬液中加入相同体积的高渗缓冲液（cpv 的 7 倍）（30 mM MOPS，250 mM 蔗糖，pH 7.2），以产生等渗环境。</li>
	<li>将匀浆转移到 10-15 mL 试管中，并在固定转子中以 1,000 × g 的速度在 4 °C 下离心 5 分钟。 注：包含未破碎细胞的原始试管可用于此目的。</li>
	<li>将含有线粒体部分的上清液转移到 1.5 mL 聚丙烯管中。</li>
	<li>可选：为了提高线粒体的产量，通过上下吹打，将步骤 1.10 中的沉淀重悬于缓冲液 A 的 7 倍细胞沉淀体积（10 mM Tris、1 mM EDTA、0.32 M 蔗糖，pH 7.4）中，并在 4 °C 下以 1,000 × g 离心 5 分钟。 在进行步骤 1.13 之前，将新的上清液与前一个上清液混合。</li>
	<li>在 4 °C 下以 16,000 × g 的微量离心机离心 2 分钟以收集线粒体粗馏分。</li>
	<li>弃去上清液，用 0.5 mL 缓冲液 A 重悬每个富含线粒体的沉淀，将两个试管的内容物合二为一，并在步骤 1.13 中描述的相同条件下离心。重复相同的过程，直到所有材料都只在一个试管中。</li>
	<li>弃去上清液，用 300 μL 缓冲液 A 重悬最终沉淀，并使用 Bradford 测定法定量线粒体蛋白浓度。像以前一样再次离心，然后继续进行第 3 部分。</li>
</ol>2. 从少量动物组织中分离线粒体
注意：具有一定置信度的应用该方案的最小组织量取决于细胞类型及其线粒体丰度，但在大多数情况下可能是 ~20-30 mg 组织。组织样品可以是新鲜材料或冷冻样品。在后一种情况下，在开始程序之前，让样品在置于冰上的均质缓冲液中解冻。
<ol><li>称重或估计尽可能接近的组织量 （mg）。</li>
	<li>用剪刀剪下组织，并在过滤器的帮助下在匀浆缓冲液中洗涤 3-4 次，注意避免丢失较小的碎片。 注意：此步骤在骨骼肌或心脏等组织中比在大脑或较软的组织中更重要，后者的细胞可以很容易地通过匀浆步骤直接解聚。</li>
	<li>添加新鲜的匀浆培养基（每克肝脏 4 mL，每克心脏、棕色脂肪组织 （BAT） 或肌肉 10 mL，以及每克大脑或肾脏 5 mL;参见 表 1 中每种情况的具体缓冲液组成）。</li>
	<li>将含有缓冲液的组织片段转移到匀浆器中，并根据所选组织遵循最佳匀浆和线粒体分离方案。</li>
	<li>从肝脏、脾脏和肾脏中分离线粒体<ol><li>在 Elvehjem-Potter 中，用电动特氟龙杵以 600 rpm 的速度进行 4 到 6 次上下冲程的均质化。</li>
		<li>将匀浆组织转移到无菌离心管中。在 4 °C 下以 1,000 × g 的水平转子离心 5 分钟。</li>
		<li>用上一步获得的上清液填充 1.5 mL 聚丙烯管。在4°C的微量离心机中以16,000 × g 离心2分钟，然后如前所述进行（步骤1.13至1.15）</li>
	</ol></li>
	<li>从心脏和肌肉样本中分离线粒体<ol><li>在 Elvehjem-Potter 中，用电动特氟龙杵以 600 rpm 的速度进行 6 到 8 次冲程的均质化。将匀浆组织转移到无菌离心管中。</li>
		<li>在 4 °C 下以 1,000 × g 的水平转子离心 5 分钟。 将上清液倒入干净的 1.5 mL 聚丙烯管中。 注：步骤 2.6.2 中获得的沉淀的第二次匀浆可以通过 4-5 次额外的冲程和 1/2 体积的匀浆缓冲液进行，以提高线粒体产量。新的上清液（在 4 °C 下再次以 1,000 × g 离心 5 分钟后）可以在进行步骤 2.6.3 之前与前一个上清液混合。提高粗线粒体产量的另一种方法是在匀浆前在胰蛋白酶溶液中解离心脏和肌肉组织样品36,37。在这种情况下，在用洋地黄皂苷溶解线粒体膜之前，必须有效地灭活/去除胰蛋白酶（步骤 3.2）。</li>
		<li>在微量离心机中以 16,000 × g 在 4 °C 下离心 2 分钟，以获得粗线粒体部分。</li>
		<li>弃去上清液，用 0.5 mL 缓冲液 AT 重悬每个富含线粒体的沉淀，将两个试管的内容物合二为一，并在步骤 2.6.3 中描述的相同条件下离心。重复相同的过程，直到所有材料都只在一个试管中。</li>
		<li>弃去上清液，用 300 μL 缓冲液 A（无 BSA）重悬最终沉淀，并使用 Bradford 测定法定量线粒体蛋白浓度。像以前一样再次离心，然后继续进行第 3 部分。</li>
	</ol></li>
	<li>从大脑中分离线粒体<ol><li>使用带有手动驱动玻璃杵的 Dounce 型玻璃均质器，以 10-15 次笔法将大脑的碎片均质化。将匀浆组织转移到无菌离心管中。</li>
		<li>在 4 °C 下以 1,000 × g 的水平转子离心 5 分钟。</li>
		<li>将上清液收集到干净的离心管中。</li>
		<li>将先前离心步骤中获得的沉淀重悬于与第一次均质中使用的相同体积的培养基 AT 中。通过重复步骤 2.7.1 中描述的过程，使用 5-10 次传递重新匀浆沉淀，并在 4 °C 下以 1,000 × g 的摆动转子离心悬浮液 5 分钟。</li>
		<li>去除上清液，将其添加到步骤 2.7.3 中制备的试管中。在固定角转子中以 10,000 × g 在 4 °C 下离心 10 分钟，以获得粗线粒体部分。</li>
		<li>弃去上清液，将沉淀重悬至培养基 AT 初始匀浆体积（步骤 2.7.1）的 1/2 中。将线粒体悬浮液分配到干净的 1.5 mL 聚丙烯管中，并在 4 °C 下在微量离心机中以 16,000 × g 离心 2 分钟。</li>
		<li>弃去上清液，用 0.5 mL 缓冲液 AT 重悬每个富含线粒体的沉淀，将两个试管的内容物合二为一，并在步骤 2.7.6 中描述的相同条件下离心。重复相同的过程，直到所有材料都只在一个试管中。</li>
		<li>弃去上清液，用 300 μL 缓冲液 A（无 BSA）重悬最终沉淀，并使用 Bradford 测定法定量线粒体蛋白浓度。像以前一样再次离心，然后继续进行第 3 部分。</li>
	</ol></li>
	<li>从 BAT 中分离线粒体<ol><li>在 Elvehjem-Potter 中，用电动特氟龙杵以 600 rpm 的速度进行 8 到 10 次上下冲程的均质化。将匀浆组织转移到无菌离心管中。</li>
		<li>在 4 °C 下以 1,000 × g 的水平转子离心 5 分钟。</li>
		<li>用上一步获得的上清液填充 1.5 mL 聚丙烯管，避免上层脂肪层。 注：可以对步骤 2.8.2 中获得的沉淀进行第二次均质化，以提高线粒体产量。新的上清液（在 4 °C 下再次以 1,000 × g 离心 5 分钟后）可以与前一个上清液混合，然后再进行步骤 2.8.4。</li>
		<li>在 4 °C 的微量离心中以 16,000 × g 离心 2 分钟，2 分钟。</li>
		<li>弃去上清液，用 0.5 mL 缓冲液 AT2 重悬每个富含线粒体的沉淀，将两个试管的内容物合二为一，并在步骤 2.8.4 中描述的相同条件下离心。重复相同的过程，直到所有材料都只在一个试管中。</li>
		<li>弃去上清液，用 300 μL 缓冲液 A（无 BSA）重悬最终沉淀，并使用 Bradford 测定法定量线粒体蛋白浓度。像以前一样再次离心，然后继续进行第 3 部分。</li>
	</ol></li>
</ol>3. Blue Native 分析样品的制备
<ol><li>在 BN 样品缓冲液（50 mM NaCl、50 mM 咪唑、5 mM 氨基己酸、4 mM PMSF）中重悬线粒体组分（从哺乳动物细胞培养物或组织中获得），以获得约 10 mg/mL 的蛋白质浓度。有关不同类型样品的预期产量，请参见 表 2 。</li>
	<li>通过添加洋地黄皂苷（10% 储备溶液）溶解线粒体膜，以获得 4 g 洋地黄皂苷/g 线粒体蛋白的比例（4 μL 洋地黄皂苷 10% 原液与 10 μL 线粒体悬浮液在步骤 3.1 中制备）。 注：必须针对每种类型的样品优化去污剂与蛋白质的比率 （g/g），以获得可重现的结果;发现 2-8 g 洋地黄皂苷/g 线粒体蛋白对大多数组织34,35 是最佳的。</li>
	<li>轻轻上下移液混合。将样品在冰上孵育 5 分钟。 注意：在此步骤之后，线粒体悬浮液应该变得更清晰（从不透明变为半透明）;否则，它可能表明去污剂的量不足以正确溶解膜。</li>
	<li>在微量离心机（约 20,000 × g ）中在 4 °C 下全速离心 25 分钟以除去不溶性物质。</li>
	<li>将上清液收集在新管中。向上清液中加入相当于初始重悬体积 1/3 的 5% G-250（考马斯蓝 G-250 5% 的 0.75 M 氨基己酸溶液）的体积（步骤 3.1，这对应于 1.6 克考马斯/g 蛋白质的最终比例）并通过移液混合。</li>
	<li>上样到凝胶上之前，请放在冰上，或在 -80 °C 下冷冻等分试样。</li>
</ol>4. Blue Native 凝胶电泳
注：电泳在冷藏室 （4-8 °C） 中进行。如果没有冷藏室设施，则可以在电泳槽中引入冷却块。使用市售的 3-13% 天然聚丙烯酰胺凝胶29，但也可以使用所需梯度浓度的自制凝胶38,39。
<ol><li>分别用冷阴极 A 和阳极缓冲液加载上腔室和下腔室。或者，将样品加载到孔中，然后小心地用阴极缓冲液 A 填充上腔室。 注：我们的实验室使用市售电泳缓冲液，但可以按照 表 1 所示制备。</li>
	<li>加载 30 至 100 μg 线粒体蛋白。 注：通常，在 10 泳道凝胶（0.5 x 0.15 cm 孔）中，每孔上样 30-50 μg 蛋白质（用于 WB）或 40-100 μg 蛋白质（用于 IGA）。阴极 A 缓冲液含有考马斯蓝 G-250，呈深蓝色，因此很难看到上样时的凝胶孔。例如，用红色标记标记每个孔的中心很方便，以帮助在此步骤中放置移液器吸头。在设置 BN-PAGE 技术时，天然分子量标记可能很有用，以确认梯度凝胶是否正确形成，以及复合物和 SCs 模式是正确的。</li>
	<li>在 80-100 V 下运行 25-30 分钟，然后在 160-180 V 下运行，将电流限制为 12 mA/凝胶，直到染料到达凝胶底部（总共 ~125-165 分钟）。</li>
	<li>如果要进行凝胶内活性测定 （IGA），当染料前沿位于凝胶中间时（电泳开始后 ~1 小时），用阴极缓冲液 B 替换阴极缓冲液 A。 注：尽管由于与 G-250 染料结合，一些条带是可见的（主要是复合物 V，可以被认为是分子量为 ~600 kDa 的“内部”制造商），因此可以在运行后记录凝胶而不进行任何染色，但通常，如果不染色或 IGA 测定，SC 和单个复合物将不可见。用于 IGA 检测或 WB 的凝胶不应染色或固定。</li>
	<li>从凝胶盒中取出凝胶，继续进行考马斯蓝染色（第 5 节）、IGA 分析（第 6 节）或 WB 免疫检测（第 7 节）。</li>
</ol>5. 凝胶染色
<ol><li>在室温 （RT） 下使用考马斯蓝染料溶液（考马斯染料 R-250，溶于 40% 甲醇、10% 乙酸中 0.25%;染色 10-15 分钟）对凝胶进行染色 10-15 分钟。</li>
	<li>在 RT 下，在 40% 甲醇和 10% 乙酸中多次洗涤（通常为 4-5 x 15 分钟）脱色。</li>
	<li>记录凝胶。</li>
</ol>6. 凝胶活性 （IGA） 测定
<ol><li>在电泳结束前准备用于分析不同呼吸复合物的 IGA 溶液，并将其保存在黑暗中（例如，通过使用深色塑料盒或用铝箔覆盖）。 表 1 中详细介绍了每种缓冲液的组成。</li>
	<li>将凝胶或要使用的泳道放在尽可能小的塑料盒中以容纳它。</li>
	<li>添加足够体积的适当溶液以覆盖凝胶（通常分别为 5 或 10 mL，用于 5 或 10 个凝胶泳道），并在室温下轻轻摇动 （60-80 rpm） 孵育，避光孵育。 注：孵育时间取决于要分析的复合物以及上样样品的性质和数量;CI 和 CIV 是最容易和最快的结果。因此，CI 活性通常会在几分钟后开始变得可见，而 CII 和 CIV 需要 ~30 分钟才能观察到，CV 需要 ~1.5-2 小时。在所有情况下，反应都可以持续数小时，并且可以通过将孵育移至冷藏室 （4-6 °C） 来降低信号增强速率，例如，进行过夜 （ON） 孵育。CIII 活性仅适用于透明的天然电泳 （CN），不适用于 BN34。</li>
	<li>当出现适当的条带后，通过去除测定溶液，用蒸馏水洗涤 2 次，并用 40% 甲醇加 10% 乙酸固定凝胶（仅用甲醇固定的 CV 除外）30 分钟来终止反应。<ol><li>要在 IGA 检测 CI 后分析 CIV 活性，请用蒸馏水洗涤凝胶 2 次，记录 CI 活性，将凝胶与 50 mM 磷酸钾缓冲液 （pH 7.2） 孵育 2 次（不固定）2 次，30 分钟，然后与完整的 CIV 反应缓冲液一起孵育，直到出现复合物 IV 条带（通常通过在室温或 4 °C 下孵育 ON）。</li>
	</ol></li>
	<li>记录凝胶。 注：对于 ATP 酶活性，由于显影条带为白色，因此在记录凝胶时，请将其放在深色背景上，以便看到透明条带。</li>
</ol>7. 蛋白质印迹分析
<ol><li>根据 表 1 准备转移缓冲液，并保持在 4 °C 直至电泳结束。</li>
	<li>将凝胶放入托盘中并加入转印缓冲液。在 RT 孵育 10-15 分钟。</li>
	<li>切下一块与凝胶大小相同的 PVDF 膜，并在搅拌下在甲醇中活化 10 秒。用蒸馏水洗涤数次并加入转印缓冲液。在 RT 孵育 10-15 分钟，轻轻摇晃。</li>
	<li>按以下顺序从下到上准备转印三明治，避免凝胶和膜之间出现气泡：盒的黑色面 - 海绵 - 吸墨纸 - 凝胶 - 膜 - 吸墨纸 - 海绵 - 盒的透明面。</li>
	<li>关闭并锁定转印盒，将三明治按正确的转印方向放入转印槽中（黑色面朝向负极）。</li>
	<li>用转移缓冲液填充转移槽，加入磁力搅拌器以搅动缓冲液，并在 80 V 下连接电源 2 小时或 100 V 下连接电源 1 小时。在 4 至 8 °C 之间（例如在冷藏室中）和传输系统中的冷却块进行传输。</li>
	<li>取回膜并继续进行标准的蛋白质印迹方案，使用针对要检测的不同呼吸复合物的特异性抗体29。</li>
</ol>

用于超复合物分析的小组织样品中的线粒体分离

<ol>
	<li>Acin-Perez, R., Fernandez-Silva, P., Peleato, M. L., Perez-Martos, A., Enriquez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+active+mitochondrial+supercomplexes.">Respiratory active mitochondrial supercomplexes.</a> Mol Cell. 32 (4), 529-539 (2008).</li><li>Schagger, H., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercomplexes+in+the+respiratory+chains+of+yeast+and+mammalian+mitochondria.">Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.</a> EMBO J. 19 (8), 1777-1783 (2000).</li><li>Schagger, H., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ratio+of+oxidative+phosphorylation+complexes+I-V+in+bovine+heart+mitochondria+and+the+composition+of+respiratory+chain+supercomplexes.">The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes.</a> J Biol Chem. 276 (41), 37861-37867 (2001).</li><li>Acin-Perez, R., Enriquez, J. 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作者声明没有利益冲突。

此处描述的方案中引入的方法学调整旨在避免损失并提高产量，同时保持线粒体复合物活性（当足够量的样品的可用性受到损害时，这一点至关重要）并复制组织或细胞系预期的 SCs 模式（参见 图 2C).为此，由于不需要高线粒体纯度来正确检测 SC，因此只要有可能，步骤、时间和体积的数量就会减少。
因此，在通过低速离心匀浆和去除细胞核和未破碎的...

超复合物 （SC） 是单个呼吸链复合物之间的超分子关联 1,2。自 Schägger 2,3 组最初鉴定 SC 并描述其组成以来，后来被其他组证实，确定它们在不同的化学计量中含有呼吸复合物 I、III 和 IV（分别为 CI、CIII 和 CIV）。可以定义两个主要的 SC 群体，一种含有 CI（单独含有 CIII 或 CIII 和 CIV）且分子量非常高（MW，较小的 SC 从 ~1.5 MDa 开始：CI + CIII2），另一种含有 CIII 和 CIV 但不含有 CIV，尺寸小得多（例如 CIII2 + CIV，具有 ~680 kDa）。这些 SC 在线粒体内膜中与游离复合物共存，比例也不同。因此，虽然 CI 主要以相关形式存在（即在 SC 中：~80% 在牛心脏中，在许多人类细胞类型中超过 90%）3，但 CIV 的游离形式非常丰富（在牛心脏中超过 80%），而 CIII 显示出更平衡的分布（~40% 在其更丰富的游离形式中， 作为二聚体，在牛心中）。
虽然它们的存在现在被普遍接受，但它们的确切作用仍在争论中 4,5,6,7,8,9,10。根据可塑性模型，根据细胞类型或代谢状态，可以存在不同比例的 SCs 和单个复合物 1,7,11。组装的这种动态性质将使细胞能够调节可用燃料的使用和氧化磷酸化 （OXPHOS） 系统的效率，以响应环境变化 4,5,7。SCs 还有助于控制活性氧的生成速率，并参与单个复合物的稳定和周转 4,12,13,14。已经描述了 SC 组装状态的改变与不同的生理和病理情况15,16 以及衰老过程17 有关。
因此，当葡萄糖被半乳糖4 取代时，酵母中 SC 模式的变化已经在酵母中被描述为2 和培养的哺乳动物细胞中的变化。空腹后小鼠肝脏8 和线粒体脂肪酸氧化被阻断时星形胶质细胞中也报道了修饰18。此外，在 Barth 综合征19、心力衰竭20、几种代谢21 和神经系统22、23、24 疾病以及不同的肿瘤25、26、27、28 中发现 SCs 和 OXPHOS 的减少或改变.在这些病理情况下，SC 组装和水平的这些改变是主要原因还是代表次要影响，仍在研究中15,16。不同的方法可以提供有关 SC 的组装和功能的信息;这些包括活性测量 8,29、超微结构分析30,31 和蛋白质组学32,33。一种越来越多地被采用的有用替代方案是 Schägger 小组为此开发的蓝色天然 （BN） 电泳34,35 的起点，这是前面提到的一些方法的起点。
这种方法需要可重现且高效的程序来获得和溶解线粒体膜，并且可以通过其他技术进行补充，例如凝胶内活性分析 （IGA）、二维电泳和蛋白质印迹 （WB）。BN 电泳对 SC 动力学的研究的一个限制可能是起始细胞或组织样品的数量。我们提出了一系列用于分析 SC 组装和功能的方案，这些方案改编自 Schägger 的小组方法，可应用于新鲜或冷冻的细胞或组织样品，起始剂量低至 20 mg 组织。

<table><tbody><tr><td>Acetic acid</td><td>PanReac</td><td>131008</td><td></td></tr><tr><td>Aminocaproic acid</td><td>Fluka Analytical</td><td>7260</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Bis Tris</td><td>Acrons Organics</td><td>327721000</td><td></td></tr><tr><td>Bradford assay</td><td>Biorad</td><td>5000002</td><td></td></tr><tr><td>Coomassie Blue G-250</td><td>Serva</td><td>17524</td><td></td></tr><tr><td>Coomassie Blue R-250</td><td>Merck</td><td>1125530025</td><td></td></tr><tr><td>Cytochrome c</td><td>Sigma-Aldrich</td><td>C2506</td><td></td></tr><tr><td>Diamino&amp;nbsp; benzidine (DAB)</td><td>Sigma-Aldrich</td><td>D5637</td><td></td></tr><tr><td>Digitonin</td><td>Sigma-Aldrich</td><td>D5628</td><td></td></tr><tr><td>EDTA</td><td>PanReac</td><td>131669</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Fatty acids free BSA</td><td>Roche</td><td>10775835001</td><td></td></tr><tr><td>Glycine</td><td>PanReac</td><td>A1067</td><td></td></tr><tr><td>Homogenizer Teflon pestle</td><td>Deltalab</td><td>196102</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac</td><td>121512</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac</td><td>121509</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich</td><td>M4125</td><td></td></tr><tr><td>Methanol</td><td>Labkem</td><td>MTOL-P0P</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac</td><td>131404</td><td></td></tr><tr><td>Mini Trans-Blot Cell</td><td>BioRad</td><td>1703930</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td></td></tr><tr><td>MTCO1 Monoclonal Antibody</td><td>Invitrogen</td><td>459600</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NADH</td><td>Roche</td><td>10107735001</td><td></td></tr><tr><td>NativePAGE 3 to 12% Mini Protein Gels</td><td>Invitrogen</td><td>BN1001BOX</td><td></td></tr><tr><td>NativePAGE Cathode Buffer Additive (20x)</td><td>Invitrogen</td><td>BN2002</td><td></td></tr><tr><td>NativePAGE Running Buffer (20x)&amp;nbsp;</td><td>Invitrogen</td><td>BN2001</td><td></td></tr><tr><td>NDUFA9 Monoclonal Antibody</td><td>Invitrogen</td><td>459100</td><td></td></tr><tr><td>Nitroblue tetrazolium salt (NBT)</td><td>Sigma-Aldrich</td><td>N6876</td><td></td></tr><tr><td>Pb(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>228621</td><td></td></tr><tr><td>PDVF Membrane</td><td>Amersham</td><td>10600023</td><td></td></tr><tr><td>Phenazine methasulfate (PMS)</td><td>Sigma-Aldrich</td><td>P9625</td><td></td></tr><tr><td>Pierce ECL Substrate</td><td>Thermo Scientific</td><td>32106</td><td></td></tr><tr><td>PMSF</td><td>Merck</td><td>PMSF-RO</td><td></td></tr><tr><td>SDHA Monoclonal Antibody</td><td>Invitrogen</td><td>459200</td><td></td></tr><tr><td>Sodium succinate</td><td>Sigma-Aldrich</td><td>S2378</td><td></td></tr><tr><td>Streptomycin/penicillin</td><td>PAN biotech</td><td>P06-07100</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S3089</td><td></td></tr><tr><td>Tris</td><td>PanReac</td><td>A2264</td><td></td></tr><tr><td>UQCRC1 Monoclonal Antibody</td><td>Invitrogen</td><td>459140</td><td></td></tr><tr><td>XCell&amp;nbsp;SureLock Mini-Cell</td><td>Invitrogen</td><td>&amp;nbsp;EI0001</td><td></td></tr></tbody></table>

isolation of mitochondria for mitochondrial supercomplex analysis from small tissue and cell culture samples

在过去的几十年里，关于呼吸超复合物 （SCs） 存在的证据积累改变了我们对线粒体电子传递链组织的理解，从而产生了“可塑性模型”的提出。该模型假设不同比例的 SCs 和复合物共存，具体取决于组织或细胞代谢状态。SCs 中组装的动态性质将使电池能够优化可用燃料的使用和电子转移的效率，最大限度地减少活性氧的产生并有利于电池适应环境变化的能力。 
最近，在神经退行性疾病（阿尔茨海默病和帕金森病）、巴斯综合征、Leigh 综合征或癌症等不同疾病中报道了 SC 组装异常。SC 组装改变在疾病进展中的作用仍需确认。然而，获得足够数量的样品来确定 SC 组装状态通常是一个挑战。这种情况发生在活检或组织样本较小或必须分开进行多次分析、细胞培养物生长缓慢或来自微流控设备、一些原代培养物或稀有细胞，或者必须分析特定昂贵处理的效果（使用纳米颗粒、非常昂贵的化合物等）。在这些情况下，需要一种有效且易于应用的方法。本文提出了一种适用于从少量细胞或组织获得富集线粒体组分的方法，以通过天然电泳然后进行凝胶内活性测定或蛋白质印迹来分析线粒体 SCs 的结构和功能。

按照上述方案获得的线粒体产量取决于多种因素，例如细胞系或组织类型、样品的性质（即，是否使用新鲜或冷冻组织）或均质化过程的效率。 表 2 中收集了来自不同细胞系和组织的线粒体的预期产量。获得线粒体组分后，下一步是呼吸 SCs 模式的分析，这是在粗线粒体样品溶解和通过 BN-PAGE 电泳分离后进行的，然后是 IGA 分析或 WB 免疫检测。 图 1 显示了培养?...

Read this Scientific Article Protocol about Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples at JoVE.com

作者聚焦：揭示氧化磷酸化系统动力学和线粒体在健康和疾病中的作用

该方案描述了一种在只有少量样品可用时分析呼吸超复合物的技术。

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析 

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

我们的研究重点是更好地了解线粒体生物发生，特别是 OXPHOS 系统的组织和调节，以及定义线粒体在从癌症到神经退行性疾病的疾病中的作用。线粒体在细胞中发挥着多种重要作用，调节其整个新陈代谢和生存。我们正在慢慢增加对线粒体复杂性及其对健康和疾病中细胞适应机制的贡献的了解。我们为 OXPHOS 系统建立了一个新的组织模型做出了贡献，该可塑性模型整合了以前的建议，并假设超级复合物和游离复合物以不同比例共存，以优化其功能并允许细胞适应。我们的方案允许从小组织或细胞培养样品开始，以获得足够的线粒体蛋白量，以通过蓝色天然电泳或其他技术分析超级复合物组装和状态和功能。我们可以使用简单的设备在短时间内以相对较低的成本重复地做到这一点。

isolation-mitochondria-for-mitochondrial-supercomplex-analysis-from

Research

JoVE Journal

Biochemistry

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

965 次观看.  University of Zaragoza. 我们的研究重点是更好地了解线粒体生物发生，特别是 OXPHOS 系统的组织和调节，以及定义线粒体在从癌症到神经退行性疾病的疾病中的作用。线粒体在细胞中发挥着多种重要作用，调节其整个新陈代谢和生存。我们正在慢慢增加对线粒体复杂性及其对健康和疾病中细胞适应机制的贡献的了解。我们为 OXPHOS 系统建立了一个新的组织模型做出了贡献，该可塑性模型整合了...

视频: 作者聚焦：揭示氧化磷酸化系统动力学和线粒体在健康和疾病中的作用

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

科研

生物化学

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

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 ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

医学

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

<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 ...

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

副本

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此视频中的章节

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

拟南芥线粒体的分离和呼吸测量

HEK 细胞 Mitoribosome 的快速分离

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

从啮齿动物心脏组织中稳健地分离线粒体

从啮齿动物心脏组织中稳健地分离线粒体

从啮齿动物心脏组织中稳健地分离线粒体