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。

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盐酸洗涤所有设备，然后用去离子水二氧化氯大量冲洗。 <ol><li> 制备 <ol><li>在4℃下制备由25mM咪唑，pH 7.0组成的阳极缓冲液;储存于4°C。 </li><li>为CN或BN PAGE准备阴极缓冲液。<ol><li>对于CN PAGE，使用7.5mM咪唑和50mM tricine;每升缓冲液中加入0.5g脱氧胆酸钠和0.2g月桂基麦芽糖苷。在4℃将pH调节至7.0，并储存在4℃。 </li><li>对于BN PAGE，使用7.5mM咪唑和50mM三羟甲基胺，并在4℃下将pH调节至7.0。储存于4°C。 </li><li>对于浅蓝色BN阴极缓冲液，使用7.5mM咪唑和50mM三氯甲烷。在4℃下将pH调节至7.0，并加入每升缓冲液20毫克考马斯。储存于4°C。 </li></ol></li><li>用50mM NaCl，50mM咪唑，2mM氨基己酸和1mM EDTA制备提取缓冲液（EB）。在4℃将pH调节至7.0，并储存在4℃。 </li><li>用75 mM咪唑和1.5M氨基己酸制备3x凝胶缓冲液（用于BN和CN凝胶）。在4℃将pH调节至7.0，并储存在4℃。 </li><li>制备0.01g Ponceau S的负载缓冲液（LB），5g甘油和5mL H 2 O.储存在室温度。 </li><li>通过向1mL 500mM 6-氨基己酸中加入50mg考马斯制备考马斯蓝。在4℃将pH调节至7.0，并储存在4℃。 </li><li>在H 2 O中制备20mM和200mM月桂基麦芽糖苷。制备200μL等分试样并将其冷冻储存。使用前解冻洗涤剂。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"></td><td colspan="2"> 3％至8％（迷你） </td><td colspan="2"> 4％至10％（最大） </td></tr><tr><td> 0.5凝胶（光） </td><td> 0.5克（重） </td><td> 0.5凝胶（光） </td><td> 0.5克（重） </td></tr><tr><td> AAB（mL） </td><td> 0.42 </td><td> 1.3 </td><td> 2.5 </td><td> 7.7 </td></tr><tr><td> CN / BN缓冲液（mL） </td><td> 1.6 </td><td> 1.6 </td><td> 8.5 </td><td> 8.5 </td></tr><tr><td> H 2 O（mL） </td><td> 2.7 </td><td> 1.4 </td><td> 14 </td><td> 6.3 </td></tr><tr><td> 甘油（g） </td><td> 0 </td><td> 0.47 </td><td> 0 </td><td> 2.5 </td></tr><tr><td> 体积（mL） </td><td> 4.72 </td><td> 4.77 </td><td> 25 </td><td> 25 </td></tr><tr><td> APS（μL） </td><td> 27 </td><td> 27 </td><td> 65 </td><td> 65 </td></tr><tr><td> TEMED（μL） </td><td> 4 </td><td> 4 </td><td> 10 </td><td> 10 </td></tr></tbody></table> 表1:需要倾倒1 Mini或Maxi-PAGE的成分的量。该表中使用的体积计算为1 mini-或1 maxi-gel，1.5 mm厚。 AAB的体积基于40％的库存解决方案。轻和重指的是浓缩物AAB离子。在梯度混合器的每一列填充有AAB溶液后，加入APS和TEMED。 <ol start="2"><li> 倾吐和运行凝胶 注意:分别为CN或BN凝胶使用3-8％或4-10％丙烯酰胺/双丙烯酰胺（AAB）梯度。 表1总结了用于微型凝胶（85mm宽×73mm高×1.5mm厚）或最大凝胶（160）的缓冲液，AAB，H 2 O，甘油，硫酸铵（APS）和四甲基乙二胺（TEMED）宽×200mm高×1.5mm厚）。具有CN或BN凝胶的玻璃板组件可以用几毫升1x凝胶缓冲液的袋子冷藏，或者用浸有1x凝胶缓冲液的纸巾包裹在一起存放。凝胶稳定使用长达一周。 <ol><li>要倾倒凝胶，将梯度混合器放置在升高的搅拌板上，以确保凝胶通过重力流动到制备的凝胶室中。 </li><li>将梯度混合器的流出室填入4.77 / 25mL（迷你/最大）的重溶液（具有较高浓度的AAB）。 </li><li>轻轻打开重型和轻型室之间的止动螺栓连接，并允许一滴溶液穿过另一侧。 注意:这从连接管和止动阀推动气泡，这将防止两个腔室之间的流动。如果双方已经被填补，这是不能做到的，因为同等的压力会阻止泡沫的流动。 </li><li>用梯度混合器的另一个室填充4.72 / 25mL（mini / maxi）的光溶液。 </li><li>将搅拌棒用重溶液放在流出室中，开始搅拌。使用不会引起冒泡的搅拌棒速度。 </li><li>快速添加APS和TEMED到每个室以启动聚合。 </li><li>打开梯度混合器的两个腔室之间的连接，并允许混合几秒钟，然后打开流出室以倒出凝胶。 注意:重力wi将两个腔室均匀地排出，并将光混入重溶液将缓慢降低凝胶从底部到顶部的丙烯酰胺密度。使用梯度搅拌机中的全部内容倒入凝胶。 <ol><li>最后，小心地安装梳子，凝胶内部的孔子可以避免气泡和层混合。 </li></ol></li><li>立即用乙醇冲洗梯度搅拌器以冲洗任何凝胶。用水冲洗并倒入第二个凝胶。让凝胶聚合​​（通常需要少于20分钟的迷你凝胶）。 </li><li>运行凝胶，将它们安装到电极组件夹具中，并用CN或浅蓝色BN阴极缓冲液填充中心/上部腔室。在将阳极缓冲液添加到外部/下部室之前，请等待几分钟以检查泄漏。 <ol><li>使用注射器或移液管，用阴极缓冲液轻轻拉出孔梳并清洗孔。 </li><li>在冷藏室（4°C）中运行凝胶或完全装入冰中。<ol><li>对于CN mini-PAGE，第一小时使用100 V，直到完成200 V，通常需要1-1.5 h。或者，在30-40V运行CN迷你页面过夜。 注意:这里的重点是高分子量蛋白质复合物，因此分子量小于140 kDa的蛋白质复合物将从凝胶中流出。电泳运行时间较短可用于保留低分子量复合物。 </li><li>对于BN maxi-PAGE，使用100 V并运行凝胶过夜（约18 h）。 注意:电流将非常低（&lt;15 mA），因此需要能够处理这些条件的电源。此时，凝胶可用于IGA或免疫印迹。在某些情况下，可以使用玻璃板上的剃须刀片将胶带或泳道从凝胶中切出，以进行电洗脱。 </li></ol></li></ol></li></ol></li><li> 样品制备 注意:膜结合的线粒体超复合物必须从th提取e内线粒体膜。要保留线粒体超复合物，请使用新鲜分离的线粒体或已经冷冻和解冻一次的样品。以下的计算/体积是给予迷你凝胶（凝胶中的10孔梳，1.5毫米厚的孔保存高达35-40μL）和最大凝胶（15孔梳子的孔可以达到200μL）。另外，将每个样品（通常为10μL）的等分试样保存在变性SDS凝胶上，以检测电压依赖性阴离子通道（VDAC）作为加载对照。 <ol><li>在微管中放置适量的量（ 例如， 10微克的微凝胶蛋白质和50-200微克最大分离的线粒体或组织匀浆），并在4℃下以17,000×g离心10-15分钟。 注意:该步骤去除一些可溶性线粒体基质和/或胞质蛋白。 </li><li>吸出并丢弃上清液，加入提取缓冲液至所需量加载到凝胶上。轻轻地将沉积物重新悬浮在冰上。如果需要，在这一点添加一般的蛋白酶抑制剂混合物。 注意:根据这里使用的设备，微型凝胶使用30μL，maxi-gel使用100μL，将蛋白质/缓冲液比例限制在不超过2μg蛋白质的1μL缓冲液中。 </li><li>添加洗涤剂（ 例如， 2μg月桂基麦芽糖苷/ 1μg蛋白质;详见代表性结果和讨论）。 注意:通常使用月桂基麦芽糖苷，但也可以使用雌二醇。 </li><li>在冰上孵育20分钟。通过研磨和/或搅拌管子在孵育期间开始轻轻混合。 </li><li>在4℃下以17,000xg离心10分钟以除去任何膜和组织碎片。 </li><li>将上清液转移到新管中。对于CN样品，每10μL样品体积加入1μLLB;样品的总体积应为app微型凝胶约40μL，maxi-gel为130μL。对于BN样品，将考马斯添加到样品中，使染料与洗涤剂的比例为1:4（w / w）。 </li><li>将30和120μL样品分别装入mini-或maxi-gel的孔中。使用每个样品的其余10μL进行变性SDS凝胶检测VDAC作为加载对照。 </li><li>为了制备分子量标记物，在60μL的BN / CN凝胶缓冲液中溶解1瓶高分子量校准组合物（参见材料表），加入120μL的H 2 O和20μL的磅。每泳道加载15μL。 </li><li>按照步骤1.2.9.2中的说明运行凝胶。 </li></ol></li></ol> 2. Cx-I和Cx-V的凝胶内测定注意:测定在室温下进行。拍摄照片，扫描或图像的开发乐队的文件。 （重要）蛋白质不能转移o硝酸纤维素膜完成IGA后。 <ol><li> Cx-I测定 <ol><li>制备。 <ol><li>制备pH为7.4的H 2 O中5mM Tris的测定缓冲液;在室温下储存。 </li><li>将10mg NADH溶于1 mL测定缓冲液中。在-20°C下保存100μL等分试样，直至使用;避免冻融。 </li><li>称取25毫克的硝基蓝四唑进入微管。 </li><li>通过在95 mL H 2 O中稀释5 mL乙酸来制备固定剂;在室温下储存。 </li></ol></li><li>进行测定<ol><li>将10 mL测定缓冲液与25 mg Nitroblue四唑（终浓度:2.5 mg / mL）和100μL10mg / mL NADH（0.1 mg / mL）混合。将其添加到从CN凝胶切除的整个凝胶，泳道或兴趣区域。 注意:这可以在透明的塑料或玻璃容器中完成。请注意，此检测不能在BN PAGE后进行。 </li><li>温和搅拌（摇杆）后，跟随蓝带的发展&gt; 3分钟。 </li><li>将凝胶固定在10-20mL乙酸溶液中，或在5mM Tris，pH 7.4中洗涤以终止反应。拍照以获取文件（参见材料表）。 </li></ol></li></ol></li><li> Cx-V测定 注意:Cx-V测定应使用重复的CN或BN凝胶进行，其中一个与5μg/ mL的低密度霉素（Cx-V抑制剂）一起孵育以显示Cx-V独立的活性。 <ol><li>制备。 <ol><li>用35mM Tris和270mM甘氨酸制备测定缓冲液;在室温下将pH调节至8.3。将缓冲液以50 mL等分试样储存，但在冷冻和解冻后检查pH值。 </li><li>在H 2 O中制备1 M MgSO4;储存于4°C直至使用。 </li><li>将27.28mg的Pb（NO 3 ） 2称量成微管。 </li><li>称量60毫克ATP进入微管。 </li><li>溶解1毫克o在1mL乙醇中的霉菌素。储存于-20°C直至使用。 </li><li>通过将50mL甲醇与50mL H 2 O混合来制备固定剂;在室温下储存。 </li></ol></li><li>进行测定<ol><li>在10-20mL测定缓冲液±5μg/ mL的寡霉素（50-100μL的1mg / mL）中，用BN或CN凝胶孵育2小时的凝胶，泳道或感兴趣区域2小时， mL在乙醇中）。 </li><li>孵育后，用14mL新鲜的测定缓冲液代替缓冲液，依次加入190μL1M MgSO 4 （14mM），27.28mg Pb（NO 3 ） 2 （5mM），60mg ATP 8mM）和75μL的寡霉素（必要时）。 </li><li>温和搅拌（摇杆）孵育并观察白色沉淀，因为寡霉素处理过的凝胶上的条带将产生非Cx-V依赖性条带。 注意:沉淀物的外观可能需要几个小时。 </li><li>将凝胶固定在甲醇 -（ 例如， 50％甲醇），因为酸性溶液会溶解铅沉淀物。拍照结果。 注意:铅沉淀物不会干扰凝胶的考马斯染色。 </li></ol></li></ol></li></ol>蛋白质转移到硝酸纤维素或聚偏二氟乙烯（PVDF）膜<ol><li> 制备25mM Tris和200mM甘氨酸的转移缓冲液。将pH调节至8.3，加入0.0005g / L SDS和200mL / L甲醇。在室温下储存。 <ol><li>用剃须刀或剪刀将硝酸纤维素或PVDF膜（孔径:0.45μm）切割成略大于凝胶的尺寸。 注意:硝化纤维素膜允许Ponceau S染色来评估蛋白质负载，而PVDF膜产生更明确的带。 </li></ol></li><li> 协议。 <ol><li>浸泡硝酸纤维素膜，滤纸和海绵在转移缓冲液中至少10分钟。将PVDF膜置于100％甲醇中15秒或根据制造商的建议将其置于转移缓冲液中。将转移套件的打开的盒子放入带有转移缓冲器的平坦的碗中。 </li><li>将海绵和1-2层滤纸放在盒背面。去除气泡。 </li><li>将凝胶放在滤纸上。通过剪切凝胶和/或膜的角来指示凝胶的取向。 </li><li>将膜放在凝胶上。去除所有气泡。 </li><li>将滤纸和海绵放在膜的顶部。清除这个三明治中的所有气泡。 </li><li>关闭纸盒并将其放入转印盒的盒式支架中。 </li><li>将蛋白质在25 V下转移约12-18小时。 </li></ol></li></ol> 4.免疫印迹<ol><li> 制备。 <ol><li>通过加入25毫升来制备一种Ponceau染色剂（500毫升）的乙酸和0.5g的Ponceau S至475mL的H 2 O;在室温下储存（可重复使用）。 </li><li>用200mM NaCl，25mM Tris-Base和2.7mM KCl制备Tris缓冲盐水（TBS）。将pH调节至8.0并储存在室温下。 </li><li>通过向TBS中加入0.5mL / L吐温20制备TBS-吐温（TBST）;在室温下储存。 </li><li>通过将5克牛奶固体溶解在100毫升TBST中来制备牛奶固体/ TBST。储存于4°C，3天内使用。 </li><li>通过将3g牛血清白蛋白（BSA，级分V）溶解在100mL TBST中来制备BSA / TBST。储存于4°C，3天内使用。 </li></ol></li><li> 协议。 <ol><li>转移完成后，将膜置于Ponceau S溶液中以显现所有转移的蛋白质。用铅笔标记膜上标记的位置，并通过照片或扫描记录Ponceau-S染色的膜。 </li><li>每次洗涤膜3次，每次10分钟TBS在温和的搅动下。 </li><li>用乳固体/ TBST在室温下封闭膜1-2小时或在温和搅拌的冷室中过夜。 </li><li>在温和搅拌下用TBST洗涤膜10分钟。 </li><li>在温和搅拌下，在冷室中与初级抗体孵育过夜。在BSA / TBST中稀释抗体（ 例如，抗ATP5A和-NDUFB6为1:1,000）。 注意:当孵育过夜时，大多数抗体在来自天然凝胶的膜上产生更好的信号。 </li><li>在温和搅拌下用TBST洗涤膜10分钟。 </li><li>在室温下温和搅拌下与二抗孵育至少60分钟。在牛奶固体/ TBST中稀释抗体（1:5000至1:50000）。 </li><li>在室温下用TBST轻轻搅拌将膜洗涤3次10分钟。 </li><li>在最后一次洗涤过程中，根据制造商的说明准备增强的化学发光（ECL）底物cturer。 </li><li>使用制造商提供的说明使用ECL底物孵育膜。 </li><li>使用ECL基板制造商提供的说明检测胶片上的信号。 </li></ol></li></ol>电解<ol><li> 制备。 <ol><li>制备25mM三羟甲基纤维素，3.75mM咪唑（4℃pH 7.0）和5mM 6-氨基己酸的洗脱缓冲液。 注意:在处理电子涂层和膜帽时，请始终戴上手套，以防止外部蛋白质受到污染。 </li></ol></li><li> 在使用电凝器进行原生电洗脱前一天，执行以下操作: <ol><li>在60℃下将洗脱缓冲液中的膜帽（截止值:3.5kDa）浸泡1小时。将盖子转移到新鲜的洗脱缓冲液中，并将其浸泡在冰箱中12小时以上。 注意:截止分子量为3.5 kDa可防止小p的损失可能容易从感兴趣的蛋白质复合物解离的蛋白质。 </li><li>用乙醇彻底清洗electroeluter模块，玻璃管，罐和盖子。用水冲洗并让设备干燥。 </li></ol></li><li> 在当地电洗脱当天，请执行以下操作。 <ol><li>将玻璃料放在要使用的每个玻璃管的底部（磨砂）中。如有必要，将玻璃管置于洗脱缓冲液中，并将玻璃料从管内向内推。 </li><li>将带有玻璃料的玻璃管推入电熨烫机的模块中。用洗脱缓冲液润湿垫圈，并将玻璃管滑入适当位置。确保玻璃管的顶部与护环平齐。 </li><li>用塞子关闭空的索环。 </li><li>在硅胶适配器的底部放置湿膜盖，并将适配器与洗脱缓冲液填充。缓慢地将适配器中的缓冲液上下移动以除去透析膜周围的任何气泡/ LI&gt; <li>将缓冲填充的适配器滑入玻璃管的底部。清除玻璃管内玻璃料上出现的所有气泡。 </li><li>每个玻璃管填充洗脱缓冲液。 </li><li>将BN PAGE的切除带放入玻璃管中（见步骤1.2.9.3）。将大块切成小块。确保玻璃管内的填充高度约为1厘米。 </li><li>将整个模块放入罐中。 </li><li>向罐中加入约600 mL冷洗脱缓冲液。确保硅适配器帽位于缓冲液中以防止透析膜上的气泡。 </li><li>在水箱的底部放置搅拌棒。 注意:搅拌会阻止气泡粘在透析膜的底部。 </li><li>在一个寒冷的房间里，以350V的速度洗脱蛋白质4小时。 </li><li>洗脱完成后，从缓冲罐中取出电凝器模块并将其放入水槽或碗中。 </li><li>如果使用塞子，将其取下在上缓冲室中。否则，使用大的移液器去除缓冲区。 </li><li>从每个玻璃管中取出缓冲液并丢弃。确保硅胶适配器保持在适当位置，并且玻璃料下方的液体不会受到干扰或晃动。 </li><li>小心地将硅胶适配器与玻璃管底部的膜帽一起取出。将硅胶盖的内容物（约400μL）吸入微管中。再加入200μL洗脱缓冲液，冲洗硅胶盖并加入微管。对所有玻璃管重复。 </li><li>当膜帽可以重复使用时，通过将膜放置在含有0.5mg / mL叠氮化钠的洗脱缓冲液中来保护膜盖。冷藏 注意:洗脱液的蛋白质浓度低，可以使用离心过滤装置进行浓缩。 </li></ol></li></ol>

<ol>
	<li>Lenaz, G., Genova, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+organisation+of+the+mitochondrial+respiratory+chain:+a+new+challenge+for+the+mechanism+and+control+of+oxidative+phosphorylation.">Supramolecular organisation of the mitochondrial respiratory chain: a new challenge for the mechanism and control of oxidative phosphorylation.</a> Adv Exp Med Biol. 748, 107-144 (2012).</li><li>Saks, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Energetic+Units+regulate+metabolism+in+cardiac+cells.">Intracellular Energetic Units regulate metabolism in cardiac cells.</a> J Mol Cell Cardiol. 52 (2), 419-436 (2012).</li><li>Schagger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal Biochem. 217 (2), 220-230 (1994).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+electrophoretic+techniques+to+identify+protein-protein+interactions.">Native electrophoretic techniques to identify protein-protein interactions.</a> Proteomics. 9 (23), 5214-5223 (2009).</li><li>Genova, M. L., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+mitochondrial+respiratory+supercomplexes.">Functional role of mitochondrial respiratory supercomplexes.</a> Biochim Biophys Acta. 1837 (4), 427-443 (2014).</li><li>Hahn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+Complete+ATP+Synthase+Dimer+Reveals+the+Molecular+Basis+of+Inner+Mitochondrial+Membrane+Morphology.">Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology.</a> Molecular cell. 63 (3), 445-456 (2016).</li><li>Strauss, M., Hofhaus, G., Schroder, R. R., Kuhlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimer+ribbons+of+ATP+synthase+shape+the+inner+mitochondrial+membrane.">Dimer ribbons of ATP synthase shape the inner mitochondrial membrane.</a> EMBO J. 27 (7), 1154-1160 (2008).</li><li>Beutner, G., Eliseev, R. A., Porter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+electron+transport+chain+activity+in+the+embryonic+heart+coincides+with+the+activation+of+mitochondrial+complex+1+and+the+formation+of+supercomplexes.">Initiation of electron transport chain activity in the embryonic heart coincides with the activation of mitochondrial complex 1 and the formation of supercomplexes.</a> PloS one. 9 (11), e113330 (2014).</li><li>Genova, M. L., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Interplay+Between+Respiratory+Supercomplexes+and+ROS+in+Aging.">The Interplay Between Respiratory Supercomplexes and ROS in Aging.</a> Antioxid Redox Signal. 23 (3), 208-238 (2015).</li><li>Rosca, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mitochondria+in+heart+failure:+decrease+in+respirasomes+and+oxidative+phosphorylation.">Cardiac mitochondria in heart failure: decrease in respirasomes and oxidative phosphorylation.</a> Cardiovasc Res. 80 (1), 30-39 (2008).</li><li>Kuter, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+within+mitochondrial+oxidative+phosphorylation+supercomplexes+and+membrane+viscosity+during+degeneration+of+dopaminergic+neurons+in+an+animal+model+of+early+Parkinson's+disease.">Adaptation within mitochondrial oxidative phosphorylation supercomplexes and membrane viscosity during degeneration of dopaminergic neurons in an animal model of early Parkinson's disease.</a> Biochim Biophys Acta. 1862 (4), 741-753 (2016).</li><li>Wittig, I., Karas, M., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+clear+native+electrophoresis+for+in-gel+functional+assays+and+fluorescence+studies+of+membrane+protein+complexes.">High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.</a> Mol Cell Proteomics. 6 (7), 1215-1225 (2007).</li><li>Fiala, G. J., Schamel, W. W., Blumenthal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+polyacrylamide+gel+electrophoresis+(BN-PAGE)+for+analysis+of+multiprotein+complexes+from+cellular+lysates.">Blue native polyacrylamide gel electrophoresis (BN-PAGE) for analysis of multiprotein complexes from cellular lysates.</a> J Vis Exp. (48), (2011).</li><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> Molecular cell. 32 (4), 529-539 (2008).</li><li>Dudkina, N. V., Eubel, H., Keegstra, W., Boekema, E. J., Braun, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+mitochondrial+supercomplex+formed+by+respiratory-chain+complexes+I+and+III.">Structure of a mitochondrial supercomplex formed by respiratory-chain complexes I and III.</a> Proc Nat Acad Sci USA. 102 (9), 3225-3229 (2005).</li><li>Giorgio, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimers+of+mitochondrial+ATP+synthase+form+the+permeability+transition+pore.">Dimers of mitochondrial ATP synthase form the permeability transition pore.</a> Proc Nat Acad Sci USA. 110 (15), 5887-5892 (2013).</li><li>Beutner, G., Sharma, V. K., Giovannucci, D. R., Yule, D. I., Sheu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+ryanodine+receptor+in+rat+heart+mitochondria.">Identification of a ryanodine receptor in rat heart mitochondria.</a> J Biol Chem. 276 (24), 21482-21488 (2001).</li><li>Rehncrona, S., Mela, L., Siesjo, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+brain+mitochondrial+function+in+the+rat+after+complete+and+incomplete+cerebral+ischemia.">Recovery of brain mitochondrial function in the rat after complete and incomplete cerebral ischemia.</a> Stroke. 10 (4), 437-446 (1979).</li><li>Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue-native+gels+to+isolate+protein+complexes+from+mitochondria.">Blue-native gels to isolate protein complexes from mitochondria.</a> Methods Cell Biol. 65, 231-244 (2001).</li><li>Althoff, T., Mills, D. J., Popot, J. L., Kuhlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrangement+of+electron+transport+chain+components+in+bovine+mitochondrial+supercomplex+I1III2IV1.">Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1.</a> EMBO J. 30 (22), 4652-4664 (2011).</li><li>Schafer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+active+mammalian+respiratory+chain+supercomplexes.">Architecture of active mammalian respiratory chain supercomplexes.</a> J Biol Chem. 281 (22), 15370-15375 (2006).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+organization+of+ATP+synthase+and+respiratory+chain+in+mitochondrial+membranes.">Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes.</a> Biochim Biophys Acta. 1787 (6), 672-680 (2009).</li><li>Davies, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+organization+of+ATP+synthase+and+complex+I+in+whole+mitochondria.">Macromolecular organization of ATP synthase and complex I in whole mitochondria.</a> Proc Nat Acad Sci USA. 108 (34), 14121-14126 (2011).</li><li>Lapuente-Brun, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercomplex+assembly+determines+electron+flux+in+the+mitochondrial+electron+transport+chain.">Supercomplex assembly determines electron flux in the mitochondrial electron transport chain.</a> Science. 340 (6140), 1567-1570 (2013).</li><li>Antonioli, P., Bachi, A., Fasoli, E., Righetti, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+removal+of+DNA+from+proteomic+samples+prior+to+two-dimensional+map+analysis.">Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis.</a> J Chromatogr A. 1216 (17), 3606-3612 (2009).</li><li>Wittig, I., Carrozzo, R., Santorelli, F. M., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assays+in+high-resolution+clear+native+gels+to+quantify+mitochondrial+complexes+in+human+biopsies+and+cell+lines.">Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.</a> Electrophoresis. 28 (21), 3811-3820 (2007).</li><li>Glancy, B., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+composition+and+function+of+red+and+white+skeletal+muscle+mitochondria.">Protein composition and function of red and white skeletal muscle mitochondria.</a> Am J Physiol Cell Physiol. 300 (6), C1280-C1290 (2011).</li><li>Wittig, I., Beckhaus, T., Wumaier, Z., Karas, M., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+estimation+of+native+proteins+by+blue+native+electrophoresis:+principles+and+practical+hints.">Mass estimation of native proteins by blue native electrophoresis: principles and practical hints.</a> Mol Cell Proteomics. 9 (10), 2149-2161 (2010).</li><li>Alavian, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+uncoupling+channel+within+the+c-subunit+ring+of+the+F1FO+ATP+synthase+is+the+mitochondrial+permeability+transition+pore.">An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore.</a> Proc Nat Acad Sci USA. 111 (29), 10580-10585 (2014).</li><li>Chance, B., Williams, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+enzymes+in+oxidative+phosphorylation.+IV.+The+respiratory+chain.">Respiratory enzymes in oxidative phosphorylation. IV. The respiratory chain.</a> J Biol Chem. 217 (1), 429-438 (1955).</li><li>Zickermann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology.+Mechanistic+insight+from+the+crystal+structure+of+mitochondrial+complex+I.">Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.</a> Science. 347 (6217), 44-49 (2015).</li><li>Zhu, J., Vinothkumar, K. R., Hirst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+mammalian+respiratory+complex+I.">Structure of mammalian respiratory complex I.</a> Nature. 536 (7616), 354-358 (2016).</li></ol>

作者没有什么可以披露的。

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装配的...

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&gt; 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。

<table><tbody><tr><td>Protean II mini-gel chamber</td><td>Biorad</td><td>1658004</td><td>Complete set to pour and run mini-gel electrophoresis</td></tr><tr><td>Protean XL maxi-gel</td><td>Biorad</td><td>1653189</td><td>Complete set to pour and run maxi-gel electrophoresis</td></tr><tr><td>Gradient maker, Hoefer SG15</td><td>VWR</td><td>95044-704</td><td>Pouring mini-gel gradients</td></tr><tr><td>Gradient maker, maxi-gel</td><td>VWR</td><td>GM-100</td><td>Pouring maxi-gel gradients</td></tr><tr><td>Transfer kit</td><td>Biorad</td><td>1703930</td><td>Complete set to wet transfer of proteins onto membranes</td></tr><tr><td>Electroeluter model 422</td><td>Biorad</td><td>1652976</td><td>Electroelution of proteins from native or SDS PAGES</td></tr><tr><td>Glass plates</td><td>Biorad</td><td>1653308</td><td>Short plates</td></tr><tr><td>Glass plates</td><td>Biorad</td><td>1653312</td><td>Spacer plates</td></tr><tr><td>Glass plates</td><td>Biorad</td><td>1651823</td><td>Inner plates</td></tr><tr><td>Glass plates</td><td>Biorad</td><td>1651824</td><td>Outer Plates</td></tr><tr><td>Power supply</td><td>Biorad</td><td>1645070</td><td>Power supply suitable for native electrophoresis</td></tr><tr><td>ECL-Western&amp;nbsp;</td><td>Thermo Scientific</td><td>32209</td><td>Chemolumniscense substrate</td></tr><tr><td>SuperSignal-West Dura</td><td>Thermo Scientific</td><td>34075</td><td>Enhanced chemolumniscense substrate</td></tr><tr><td>Film/autoradiography film</td><td>GE Health care</td><td>28906845</td><td>Documentation of Western blots</td></tr><tr><td>Film processor CP1000</td><td>Agfa</td><td>NC0872640</td><td></td></tr><tr><td>Canon Power Shot 640&amp;nbsp;</td><td>Canon</td><td>NA</td><td>Taking photos to document gels, membranes and blots.</td></tr><tr><td>Canon Power Shot 640 Camera hood&amp;nbsp;</td><td>Canon</td><td></td><td>shielding camera for photos being taken on a light table</td></tr><tr><td>Acrylamide/bisacrylamide</td><td>Biorad</td><td>1610148</td><td>40% pre-mixed solution</td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7403</td><td></td></tr><tr><td>SDS (sodium dodecyl sulfate)</td><td>Invitrogen</td><td>15525-017</td><td></td></tr><tr><td>Tris-base</td><td>Sigma</td><td>T1503</td><td>Buffer</td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>Sodium deoxychelate</td><td>Sigma</td><td>D66750</td><td>Detergent</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td>Buffer</td></tr><tr><td>Imidazole</td><td>Sigma</td><td>I15513</td><td>Buffer</td></tr><tr><td>Lauryl maltoside</td><td>Sigma</td><td>D4641</td><td>Detergent</td></tr><tr><td>Coomassie G250</td><td>Biorad</td><td>161-0406</td><td></td></tr><tr><td>Aminohexanoic acid</td><td>Sigma</td><td>O7260</td><td></td></tr><tr><td>Native&amp;nbsp; molecular weight kit</td><td>GE Health care&amp;nbsp;</td><td>17-0445-01</td><td>High molecular weight calibraition kit for native electrophoresis.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>NADH</td><td>Sigma</td><td>N4505</td><td></td></tr><tr><td>Nitroblue tetrazolium</td><td>Sigma</td><td>N6639</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>ATP&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>A2383</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Lead(II) nitrate (Pb(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;):</td><td>Sigma</td><td>228621</td><td></td></tr><tr><td>Oligomycin</td><td>Sigma</td><td>O4876</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P3504</td><td></td></tr><tr><td>anti-ATP5A</td><td>Abcam</td><td>ab14748</td><td>antibody to ATP synthase subunit ATP5A</td></tr><tr><td>anti-NDUFB6</td><td>Abcam</td><td>ab110244</td><td>antibody to Cx-1 subunit NDUFB6</td></tr><tr><td>anti-VDAC</td><td>Calbiochem</td><td>529534</td><td>antibody to VDAC</td></tr><tr><td>ECL HRP linked antibody</td><td>GE Health Care</td><td>NA931V</td><td>secondary antibody to ATP5A, NDUFB6 and VDAC</td></tr><tr><td>Blocking reagent</td><td>Biorad</td><td>170-6404</td><td></td></tr><tr><td>BSA</td><td></td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>potassium chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Silver staining Kit</td><td>Invitrogen</td><td>LC6070</td><td></td></tr></tbody></table>

analyzing supercomplexes of the mitochondrial electron transport chain with native electrophoresis, in-gel assays, and electroelution

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。

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?...

Watch this Scientific Journal Video about Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution at JoVE.com

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

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。

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 ...

analyzing-supercomplexes-mitochondrial-electron-transport-chain-with

Research

JoVE Journal

Biochemistry

14.0K 次观看.  University of Rochester. 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。 

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

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

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

C2C12 Myotubes 测量反式等离子体膜电子传输

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

生物学

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 ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

测量来自母猪后代的先前冷冻心脏组织中的线粒体电子转移复合物:评估运动诱导的线粒体生物能量变化的模型

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

用于超复合物分析的小组织样品中的线粒体分离

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

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

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

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

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 ...

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

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在人类胚胎干细胞的线粒体复杂的活动初探

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

测量来自母猪后代的先前冷冻心脏组织中的线粒体电子转移复合物:评估运动诱导的线粒体生物能量变化的模型

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

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

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