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
Summary
该方案描述了使用天然电泳分离功能性线粒体电子传递链复合物（Cx）IV及其超复合物，以揭示其组装和结构信息。天然凝胶可以进行免疫印迹，凝胶内测定，并通过电洗脱纯化以进一步表征单个复合物。
Abstract
线粒体电子传递链（ETC）将从各种燃料分解的能量转化为细胞的生物能量货币。 ETC由5个大型蛋白质复合物组成，它们还组装成称为呼吸面罩（CI，C-III，C-IV）和合成体（CV）的超复合物，提高电子传输和ATP生产的效率。 50多年来已经使用各种方法来测量ETC功能，但是这些协议并不提供关于单个复合体和超级复合物组装的信息。该方案描述了天然凝胶聚丙烯酰胺凝胶电泳（PAGE）的技术，该方法在20多年前被修改以研究ETC复合物结构。天然电泳可以将ETC复合物分离成其活性形式，然后可以使用免疫印迹，凝胶内测定（IGA）和电洗脱纯化来研究这些复合物。通过组合天然凝胶PAGE与其他线粒体测定结果的结果，可以获得ETC活性，动态组装和反汇编的完整图片，以及如何调节线粒体结构和功能。这项工作还将讨论这些技术的局限性。总之，以下给出的本地PAGE技术，随后的免疫印迹，IGA和电洗脱技术是研究线粒体ETC超复合物的功能和组成的有效方法。
Introduction
ATP形式的线粒体能量不仅对于细胞存活是必需的，而且对于细胞死亡的调控是重要的。通过氧化磷酸化产生ATP需要功能性电子传递链（ETC; Cx-1至IV）和线粒体ATP合成酶（Cx-V）。最近的研究表明，这些大蛋白质复合物被组织成超复合物，称为呼吸器和合成体1,2 。分析这些大型复合物和超复合物的组装，动力学和活性调节是很困难的。尽管用氧电极进行的氧消耗测量和使用分光光度计进行的酶测定可以提供关于ETC复合物活性的有价值的信息，但是这些测定不能提供关于所涉及的蛋白质复合物或超复合物的存在，大小和亚基组成的信息。然而，蓝色和清楚的本地人的发展（BN和CN）PAGE 3创造了一个强大的工具，用于揭示复杂组合和装配/拆卸的重要信息，以及生理和病理条件下这些重要呼吸复合物超分子组织的动态调节。
这些复合物组装成高阶超复合物似乎调节线粒体结构和功能5 。例如，呼吸装置组装增加了电子转移的效率和跨越线粒体内膜的质子动力的产生5 。此外，合成体的装配不仅提高了ATP生产的效率和将能量转移到细胞质2中 ，而且还将线粒体内膜模制成管状碎屑6 ，/ sup> 7 。在小鼠胚胎心脏发育过程中超复合组装的研究表明，心脏中含有Cx-1的超复合物的产生在大约胚胎期13.58开始。其他人已经表明，由于老化或缺血/再灌注损伤9或10 ，含有Cx-1的超复合物的量在心脏中降低，或者可能在神经退行性疾病进展中起作用。
该方案描述了可用于研究ETC复合物和超复合物的组装和活性的天然凝胶PAGE的方法。可以通过分离CN或BN聚丙烯酰胺凝胶中的蛋白质复合物来评估线粒体超复合物的近似分子量。 CN PAGE还允许直接在凝胶中显示所有线粒体复合物的酶活性（凝胶内测定;IGA） 12 。这项工作通过强调Cx-I通过IGA氧化NADH的能力和由于IGA的Cx-V的ATP水解活性而导致的合成体的存在，证明了呼吸面具的活性。含有Cx-1和Cx-V的多重复合物和超复合物也可以通过将蛋白质转移到硝酸纤维素膜上并进行免疫印迹来证明。这种方法的优点是BN或CN PAGE通常根据其生理大小和组成分离蛋白复合物;转移到膜保留了这种带状图案。在BN或CN PAGE中分析蛋白质复合物也可以使用2D-PAGE（参见Fiala 等人 13 ，用于论证）或通过蔗糖密度离心14,15完成 。为了进一步分析特异性条带，可以从BN PAGE中切下来，这种蛋白质复合物的蛋白质可以是纯净的d通过在天然条件下电泳。天然电洗脱可以在几个小时内进行，这可能对从凝胶到周围缓冲液中的蛋白质的被动扩散（如参考文献16中所使用的）有显着差异。
总之，这些方法描述了允许从线粒体膜进一步表征高分子量超复合物的几种方法。
Protocol
使用C57BL / 6N小鼠（野生型）的心脏进行所有实验。在颈椎脱位之前，用二氧化碳麻醉小鼠，所有手术都严格按照罗切斯特大学实验动物医学部进行，并符合州法律，联邦法规和NIH政策。该议定书由罗切斯特大学动物保护和使用委员会（动物资源大学委员会）批准。 CN和BN PAGE 注意：用于BN和CN PAGE的所有设备均不得含有洗涤剂。为了确保这一点，用0.1M盐酸?…
Representative Results
为了可视化线粒体超复合物，使用来自小鼠的新鲜分离的线粒体17,18 。线粒体超复合物对重复的冷冻和解冻循环敏感，导致其分解，尽管这可能是一些研究人员可以忍受的。如果冷藏是必要的储存，为了确保最佳结果，样品不应经受多于一个循环的冻融。 为了用BN PAGE可视化线粒体ETC复合物，将100μg来自分离?…
Discussion
功能性ETC对于线粒体ATP生成是必需的。 ETC的复合物能够形成两种类型的超复合物：呼吸机（Cx-I，-III和-IV） 1和合成体（Cx-V） 2 。每个复合体的组装对于完整的ETC是必需的，而将ETC组织成超复合物被认为可以提高总体ETC效率5,22。这些超级复合体如何组装和拆卸还不太清楚，但是这里介绍的协议可能有助于更好地了解这些过程。
研究ETC装配?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到美国心脏病协会创始人会员[12GRNT12060233]和罗切斯特大学强力儿童研究中心的资助。
Materials
| Protean II mini-gel chamber | Biorad | 1658004 | Complete set to pour and run mini-gel electrophoresis |
| Protean XL maxi-gel | Biorad | 1653189 | Complete set to pour and run maxi-gel electrophoresis |
| Gradient maker, Hoefer SG15 | VWR | 95044-704 | Pouring mini-gel gradients |
| Gradient maker, maxi-gel | VWR | GM-100 | Pouring maxi-gel gradients |
| Transfer kit | Biorad | 1703930 | Complete set to wet transfer of proteins onto membranes |
| Electroeluter model 422 | Biorad | 1652976 | Electroelution of proteins from native or SDS PAGES |
| Glass plates | Biorad | 1653308 | Short plates |
| Glass plates | Biorad | 1653312 | Spacer plates |
| Glass plates | Biorad | 1651823 | Inner plates |
| Glass plates | Biorad | 1651824 | Outer Plates |
| Power supply | Biorad | 1645070 | Power supply suitable for native electrophoresis |
| ECL-Western | Thermo Scientific | 32209 | Chemolumniscense substrate |
| SuperSignal-West Dura | Thermo Scientific | 34075 | Enhanced chemolumniscense substrate |
| Film/autoradiography film | GE Health care | 28906845 | Documentation of Western blots |
| Film processor CP1000 | Agfa | NC0872640 | |
| Canon Power Shot 640 | Canon | NA | Taking photos to document gels, membranes and blots. |
| Canon Power Shot 640 Camera hood | Canon | shielding camera for photos being taken on a light table | |
| Acrylamide/bisacrylamide | Biorad | 1610148 | 40% pre-mixed solution |
| Glycine | Sigma | G7403 | |
| SDS (sodium dodecyl sulfate) | Invitrogen | 15525-017 | |
| Tris-base | Sigma | T1503 | Buffer |
| Tricine | Sigma | T0377 | |
| Sodium deoxychelate | Sigma | D66750 | Detergent |
| EDTA | Sigma | E5134 | |
| Sucrose | Sigma | S9378 | |
| MOPS | Sigma | M1254 | Buffer |
| Imidazole | Sigma | I15513 | Buffer |
| Lauryl maltoside | Sigma | D4641 | Detergent |
| Coomassie G250 | Biorad | 161-0406 | |
| Aminohexanoic acid | Sigma | O7260 | |
| Native molecular weight kit | GE Health care | 17-0445-01 | High molecular weight calibraition kit for native electrophoresis. |
| Name | Company | Catalog Number | Comments |
| NADH | Sigma | N4505 | |
| Nitroblue tetrazolium | Sigma | N6639 | |
| Tris HCL | Sigma | T3253 | |
| ATP | Sigma | A2383 | |
| Name | Company | Catalog Number | Comments |
| Lead(II) nitrate (Pb(NO3)2): | Sigma | 228621 | |
| Oligomycin | Sigma | O4876 | |
| Name | Company | Catalog Number | Comments |
| Ponceau S | Sigma | P3504 | |
| anti-ATP5A | Abcam | ab14748 | antibody to ATP synthase subunit ATP5A |
| anti-NDUFB6 | Abcam | ab110244 | antibody to Cx-1 subunit NDUFB6 |
| anti-VDAC | Calbiochem | 529534 | antibody to VDAC |
| ECL HRP linked antibody | GE Health Care | NA931V | secondary antibody to ATP5A, NDUFB6 and VDAC |
| Blocking reagent | Biorad | 170-6404 | |
| BSA | |||
| sodium chloride | Sigma | S9888 | |
| potassium chloride | Sigma | P9541 | |
| EGTA | Sigma | E3889 | |
| Name | Company | Catalog Number | Comments |
| Silver staining Kit | Invitrogen | LC6070 |
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Citar este artigo
Beutner, G., Porter Jr., G. A. Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution. J. Vis. Exp. (124), e55738, doi:10.3791/55738 (2017).