这项研究得到了澳大利亚研究理事会植物能源生物学卓越中心 CE140100008, 澳大利亚研究理事会未来的奖学金 (FT130100112) MWM, 和费奥多 Lynen 研究奖学金 (亚历山大·冯洪堡基金会, 德国) 到 JS。

该协议用于分离的完整的线粒体从拟南芥的器官生长在土壤上使用连续的胶体密度梯度。所有程序在材料的汇集以后在4° c 执行。
1. 研磨介质、洗涤缓冲液和梯度溶液的制备
<ol><li>准备300毫升的研磨介质 (减去抗坏血酸和半胱氨酸) 和200毫升的2x 洗涤缓冲器每表 1在隔离之前的一天, 让他们在4° c 冷。 注: 将钠抗坏血酸和 l-半胱氨酸 (自由基) 添加到最终浓度为17.84 毫米和20.36 毫米, 分别为在上午的研磨介质的隔离。如果线粒体是用于西部印迹或蓝色本地-聚丙烯酰胺凝胶电泳 (BN 页) 分析, 构成2x 洗涤缓冲没有 BSA。</li>
	<li>在线粒体隔离的早晨准备渐变 (图 1和表 1)。<ol>
<li>在两个烧杯中进行重光梯度解;35毫升的每一个是足够的两个渐变管。</li>
		<li>清洗的梯度倒和 PVC 蠕动油管与去离子水的商会, 确保甚至流经油管从所有油管。</li>
		<li>放置2离心管 (50 毫升), 在一个轻微的角度, 在冰与 PVC 蠕动油管插座贴在管子的内部, 以确保梯度解决方案的一侧运行的管。</li>
		<li>关闭内部和外部室之间的连接 (黑色拉杆向下)。将梯度倒放在带有小搅拌杆的磁力搅拌器上。</li>
		<li>将35毫升重梯度溶液倒入内腔室 (带导管出口的腔室)。将重梯度溶液分配到放置在冰上的两个离心管, 直到一半的蠕动泵速率设置为快 (300 毫升/小时)。</li>
		<li>将35毫升的光梯度溶液倒入外腔 (不带油管出口的腔室)。打开商会之间的连接 (推黑杠杆上半), 并允许解决方案轻轻混合。使用磁力搅拌器混合溶液。</li>
		<li>允许解决方案运行缓慢 (60 毫升/小时), 直到所有的梯度混合已从分庭产生了两个 0-4.4% (w/v) 酮 (PVP) 渐变填充管。保持在冰上的梯度, 直到准备在步骤2.12 使用。 注: 一旦梯度已准备, 稀释2x 洗涤介质到1x 与 prechilled-去离子水和保持冷在4° c。</li>
	</ol>
</li>
</ol>2. 均匀化和线粒体分离
<ol><li>修剪整个玫瑰花组织从4周的拟南芥植物生长在土壤与剪刀, 至少80植物将需要1准备。 注:10-14 天古老的水文化, 至少5盆/准备, 或2周老苗生长在村 & Skoog 基盐混合物 (MS) 琼脂板, 至少4板, 也可以使用。</li>
	<li>在预4° c 的大型砂浆和杵中, 用75毫升研磨介质 (表 1) 将一半的植物材料切成小块, 并在几分钟内研磨, 直到没有大块的组织留下。</li>
	<li>预湿4层过滤材料 (22-25 µm 孔大小) 与研磨介质和过滤器匀浆通过4层过滤材料通过塑料漏斗成500毫升圆锥烧瓶。</li>
	<li>用另外75毫升研磨介质研磨剩余的一半的组织。</li>
	<li>将匀在过滤材料中结合, 尽可能多地匀浆。</li>
	<li>再用剩余的150毫升研磨介质研磨残留在过滤材料上的剩余组织, 再过滤到500毫升的圆锥烧瓶中。</li>
	<li>离心过滤匀浆在 8 pre-chilled 50 毫升塑料离心管在 2500 x g 在4° c 在一个固定角度转子为 5 min。</li>
	<li>将上清液倒入清洁离心管 (50 毫升; 避免扰乱绿色颗粒), 在4° c 下离心 17500 x g, 在固定角度的转子上进行20分钟。</li>
	<li>放弃所有, 但 < 3 毫升的上清通过吸入 (或轻轻地倒在不扰乱颗粒)。用小的细画笔轻轻地重残留上清液中的颗粒。</li>
	<li>在每根管子上加10毫升的1x 洗涤缓冲器, 并将4管组合成1清洁离心管 (即, 产生2管)。</li>
	<li>用1x 洗涤缓冲器将这些管子填满50毫升, 重复步骤 2.7-2.9。</li>
	<li>将粗粒的线粒体颗粒放入1管中, 用滴管均匀地分散, 用细刷 (少量的洗涤缓冲器可以使用, 但最后的体积需要小于3毫升, 以适应梯度的顶部)。轻轻地将线粒体悬浮液与滴管一起放到2连续的 0-4.4% (w/v) PVP 梯度上。</li>
	<li>平衡管重量 (使用1x 洗涤缓冲) 和离心机在 4万 x g 在4° c 为 40 min. 确保中断被关闭。线粒体会迁移到管底附近的一个波段, 或者可能出现在管子底部的重溶液中 (由一个多云、黄色的带来识别;图 2)。</li>
	<li>小心地去除线粒体带上方5厘米以上的溶液的吸入。</li>
	<li>将含有线粒体的剩余溶液分布到2干净的管子中, 并填充1x 洗涤缓冲器。均匀地分布胶体密度梯度和线粒体通过覆盖管口与塑料石蜡膜和倒置多次。离心机为15分钟在 3.1万 x g在4° c 以慢刹车。 注: 在离心前, 确保线粒体和剩余的胶体密度梯度均匀分布。否则, 胶体密度梯度可以集中在管的底部, 防止线粒体的粒化。</li>
	<li>用抽吸去除上清液, 用1x 洗涤缓冲器将管子填满50毫升, 再倒置以确保混合。离心机为15分钟在 3.1万 x g 在4° c 以慢刹车。</li>
	<li>吸入上清液, 收集线粒体小球小体积 (约500µL) 尽可能使用一个吸管与修改200µL 提示 (削减尖端略高于其年底增加其开放)。将线粒体放入一个1.5 毫升的管中, 并在-80 ° c 时保持在冰上以备立即使用或贮存。 注: 如果线粒体将用于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS 页), BN 页, 或免疫分析, 使用1x 洗涤缓冲没有 BSA 为最后的洗涤 (即, 步骤2.15 和 2.16)。</li>
	<li>用布拉德福法测定线粒体的蛋白质浓度。 注: BN-页离心机等分在4° c, 储存颗粒在-80 ° c 直到使用。对于耗氧量的测量, 只能使用新近分离的线粒体。</li>
</ol>3. 耗氧量测量
注: 新分离的植物线粒体的耗氧量可以用克拉克型氧电极进行分析, 从而确定线粒体完整性、细胞色素c通路活动和替代通路活动。
<ol><li>软件安装<ol>
<li>在用于氧气消耗量测量的计算机上安装许可的氧气监控软件。</li>
	</ol></li>
	<li>呼吸介质、基质、化学储存液和克拉克型氧电极的制备<ol>
<li>准备呼吸培养基 (300 毫米蔗糖, 10 毫米氯化钠, 5 毫米,2PO4, 2 毫米 MgSO4, 0.1% (含/五) 牛血清白蛋白 (BSA), 10 毫米的附加费 (pH 7.2)) 用于氧耗测定。</li>
		<li>准备用于测量线粒体呼吸的基底、抑制剂和效应物 (表 2)。这些可以及早准备并且存放在-20 ° c。 注: 使用氢氧化钠前, 将酸性溶液如有机酸的 ph 值与 ph 值7.0 中和。</li>
		<li>将呼吸介质加热到与试验室相同的温度 (25 ° c)。</li>
		<li>按照制造商协议中的描述组装克拉克型氧电极。</li>
		<li>地方1毫升空气水 (空气水是通过大力摇动少量的去离子水在一个大圆锥烧瓶) 在测量室。要打开搅拌器, 请单击 "搅拌器速度" 按钮。单击 "On (On)" 按钮, 将搅拌器速度设置为 65 rpm。单击 "确定" 继续。25° c 连续搅拌水。</li>
	</ol></li>
	<li>氧电极的标定<ol>
<li>将用于校准的水加热到与试验室相同的温度 (25 ° c)。</li>
		<li>等待电流稳定和校准氧电极。</li>
		<li>要开始校准氧电极, 点击 "校准" 按钮, 选择 "液相校准", 然后 "空气饱和水"。</li>
		<li>一个新的窗口将打开 (氧气校准 (液相)-步骤1的 5)。选择要校准的通道 (电极控制盒连接的通道) 并输入变量 (将 "室温" 设置为25° c, "大气压" 为101.32 帕)。单击 "确定" 继续。</li>
		<li>在下一步 (步骤2的 5), 设置搅拌器的速度 (65 rpm), 并点击 "OK" 继续。</li>
		<li>在步骤3的 5, 等待信号到达一个高原。期限 "到达的高原, 按 OK 继续" 将出现在箱子。单击 "确定" 继续。</li>
		<li>在5步4中, 通过加入5毫克钠硫酸钠在燃烧室内建立零氧。单击 "确定" 继续。 注: 氧浓度下降应在微量可见, 并应达到0。</li>
		<li>在校准的最后一步 (步骤5的 5), 等待信号到达一个高原。期限 "到达的高原, 按 OK 继续" 将出现在箱子。单击 "确定" 继续。</li>
		<li>单击 "保存校准" 以保存校准。将打开一个新窗口。单击 "确定" 以确认保存氧气控制箱的新校准。 注: 在校准成功后, "氧 (nmol/毫升)" 标签将出现在 y-axis 上。</li>
		<li>校准后, 通过冲洗腔内的水 (至少 5-10 次) 来清洁测量室, 以确保适当的清洁。将1毫升的呼吸介质放入测量室, 使膜平衡几分钟, 直到氧耗痕呈线性。</li>
		<li>适应氧气消耗量跟踪的规模, 得到一个好的斜坡景观。单击 "缩放 XY" 并输入变量 (设置 "氧气" 到 0-300 和 "时间轴" 到 0-45 min)。</li>
	</ol></li>
	<li>线粒体完整性的测定<ol>
<li>与柱塞打开, 增加970µL 空气呼吸介质到反应室。</li>
		<li>添加30µL 分离的线粒体或150µg 的总线粒体蛋白, 确定后线粒体分离 (总蛋白需要包括在计算之后) 到反应室使用吸管与切割尖端。</li>
		<li>用磁力搅拌器棒 (65 rpm) 在25° c 下连续搅拌线粒体悬浮液, air-saturate 溶液。</li>
		<li>用柱塞密封室, 点击 "开始录音" 记录耗氧量, 并允许氧气消耗轨迹稳定 (1-2 分钟)。</li>
		<li>在添加 10 mM 抗坏血酸和25µM 细胞色素c后, 手动确定耗氧率, 以便5分钟获得 "洗涤剂前" 率。 注意: 要在跟踪中直接添加衬底、抑制剂和效应物, 请单击 "加入事件标记" 按钮, 然后输入相应的标签名称。单击 "确定" 继续。</li>
		<li>在加入 0.05% (v/v) 洗涤剂后, 手动测定3分钟的耗氧率, 给出 "后洗涤剂" 的速率。单击 "停止录制"。</li>
		<li>单击 "更改表的速率" 按钮。2游标将显示为图形屏幕上的垂直线。单击并将两个速率游标拖动到跟踪中的所需位置, 以定义速率间隔。使用左速率游标沿跟踪移动速率间隔。使用正确的速率游标设置速率间隔。氧气监控软件会在两个游标之间自动绘制一条最佳匹配线, 同时沿跟踪移动速率间隔游标。</li>
		<li>在定义了所需的速率间隔和位置之后, 用右键单击2游标中的 1, 然后选择 "将速率添加到表", 将速率输入到表中。单击 "添加" 按钮以确认在主费率表上显示速率。</li>
		<li>通过将 "洗涤剂" 率除以 "洗涤剂后" 率来计算线粒体的完整性, 以百分比表示, 并将其从100中减去。</li>
	</ol></li>
	<li>线粒体呼吸的测定<ol>
<li>与柱塞打开, 增加970µL 空气呼吸介质到反应室。添加500µM ATP 和30µL 分离的线粒体或150µg 总线粒体蛋白使用吸管与切尖的呼吸介质在反应室。</li>
		<li>使用磁性搅拌器棒 (65 rpm) 在25° c 下连续搅拌线粒体悬浮液。</li>
		<li>关闭柱塞, 点击 "开始录音" 记录耗氧量, 等待 1-2 分钟的氧气消耗率稳定 (当耗氧量是线性的)。加5毫米琥珀酸。记录耗氧率约2分钟。 注意: 要在跟踪中直接添加衬底、抑制剂和效应物, 请单击 "加入事件标记" 按钮, 然后输入相应的标签名称。单击 "确定" 继续。</li>
		<li>添加1毫米二磷酸腺苷 (ADP)。继续记录耗氧率为2分钟。</li>
		<li>加1毫米 NADH。记录氧耗率为 4-8 分钟。这个速率是通过细胞色素氧化酶进行呼吸的能力。</li>
		<li>添加1毫米氰化钾 (KCN) (或2.5 µM 噻唑或5µM antimycin A) 抑制细胞色素c通路, 并继续记录约2分钟, 以观察氧气消耗通过替代氧化酶。</li>
		<li>添加5毫米糖 (德勤) 和10毫米丙酮酸, 以充分激活替代氧化酶。继续记录 5-7 分钟: 这是替代氧化酶的能力。</li>
		<li>添加500µM n-丙酸酯 (nPG), 并记录2分钟。这就评估了不能被化学抑制的剩余耗氧率。从记录的其他速率中减去此速率, 因为它不太可能是线粒体的来源。在细胞色素c和 AOX 通路 (由 KCN 和 nPG) 的抑制后仍然发生的氧消耗很可能来自过氧化物存在于孤立的线粒体9中的污染物。</li>
		<li>单击 "停止录制"。</li>
		<li>单击 "更改表的速率" 按钮, 并在两个游标中的一个上单击鼠标右键, 然后选择 "将速率添加到表中"。单击 "添加" 按钮以确认在主费率表上显示速率。 注: 当添加的效应剂溶解在有机溶剂 (e. g, antimycin, nPG, 噻唑), 室和柱塞必须用乙醇冲洗, 然后水, 以确保适当的清洁。</li>
	</ol></li>
</ol>

<ol>
	<li>Day, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highlights+in+plant+mitochondrial+research.+Methods+in+molecular+biology.">Highlights in plant mitochondrial research. Methods in molecular biology.</a> Plant mitochondria. 1305, v-xvi (2015).</li><li>Millar, A. H., Carrie, C., Pogson, B., Whelan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+function-location+nexus:+Using+multiple+lines+of+evidence+in+defining+the+subcellular+location+of+plant+proteins.">Exploring the function-location nexus: Using multiple lines of evidence in defining the subcellular location of plant proteins.</a> Plant Cell. 21 (6), 1625-1631 (2009).</li><li>Eubel, H., 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=Free-flow+electrophoresis+for+purification+of+plant+mitochondria+by+surface+charge.">Free-flow electrophoresis for purification of plant mitochondria by surface charge.</a> Plant J. 52 (3), 583-594 (2007).</li><li>Murcha, M. W., 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=Protein+import+into+plant+mitochondria:+Signals,+machinery,+processing,+and+regulation.">Protein import into plant mitochondria: Signals, machinery, processing, and regulation.</a> J. Exp. Bot. 65 (22), 6301-6335 (2014).</li><li>Carrie, C., 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=Approaches+to+defining+dual-targeted+proteins+in+Arabidopsis.">Approaches to defining dual-targeted proteins in Arabidopsis.</a> Plant J. 57 (6), 1128-1139 (2009).</li><li>Pinto, G., Radulovic, M., Godovac-Zimmermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+perspectives+in+the+redox+code+-+Mass+spectrometric+proteomics+studies+of+moonlighting+proteins.">Spatial perspectives in the redox code - Mass spectrometric proteomics studies of moonlighting proteins.</a> Mass Spectrom. Rev. , (2016).</li><li>Day, D. A., Neuburger, M., Douce, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterisation+of+chlorophyll-free+mitochondria+from+pea+leaves.">Biochemical characterisation of chlorophyll-free mitochondria from pea leaves.</a> Aust. J. Plant Physiol. 12 (3), 219-228 (1985).</li><li>Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S., Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+peptide+stable+isotope+dimethyl+labeling+for+quantitative+proteomics.">Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.</a> Nat. Protoc. 4 (4), 484-494 (2009).</li><li>Cheah, M. H., 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=Online+oxygen+kinetic+isotope+effects+using+membrane+inlet+mass+spectrometry+can+differentiate+between+oxidases+for+mechanistic+studies+and+calculation+of+their+contributions+to+oxygen+consumption+in+whole+tissues.">Online oxygen kinetic isotope effects using membrane inlet mass spectrometry can differentiate between oxidases for mechanistic studies and calculation of their contributions to oxygen consumption in whole tissues.</a> Anal Chem. 86 (10), 5171-5178 (2014).</li><li>Chrobok, D., 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=Dissecting+the+metabolic+role+of+mitochondria+during+developmental+leaf+senescence.">Dissecting the metabolic role of mitochondria during developmental leaf senescence.</a> Plant Physiol. 172 (4), 2132-2153 (2016).</li><li>Lee, C. P., Eubel, H., O'Toole, N., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+proteomics+of+root+and+shoot+mitochondria+and+transcript+analysis+to+define+constitutive+and+variable+components+in+plant+mitochondria.">Combining proteomics of root and shoot mitochondria and transcript analysis to define constitutive and variable components in plant mitochondria.</a> Phytochemistry. 72 (10), 1092-1098 (2011).</li><li>Lee, C. P., Eubel, H., Solheim, C., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+proteome+heterogeneity+between+tissues+from+the+vegetative+and+reproductive+stages+of+Arabidopsis+thaliana+development.">Mitochondrial proteome heterogeneity between tissues from the vegetative and reproductive stages of Arabidopsis thaliana development.</a> J. Proteome Res. 11 (6), 3326-3343 (2012).</li><li>Millar, A. H., Whelan, J., Soole, K. L., Day, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+regulation+of+mitochondrial+respiration+in+plants.">Organization and regulation of mitochondrial respiration in plants.</a> Annu. Rev. Plant Biol. 62, 79-104 (2011).</li><li>Peters, 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=Complex+I+-+complex+II+ratio+strongly+differs+in+various+organs+of+Arabidopsis+thaliana.">Complex I - complex II ratio strongly differs in various organs of Arabidopsis thaliana.</a> Plant Mol. Biol. 79 (3), 273-284 (2012).</li><li>Werhahn, W. H., 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=Purification+and+characterization+of+the+preprotein+translocase+of+the+outer+mitochondrial+membrane+from+Arabidopsis+thaliana:+Identification+of+multiple+forms+of+TOM20.">Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis thaliana: Identification of multiple forms of TOM20.</a> Plant Physiol. 125 (2), 943-954 (2001).</li><li>Heazlewood, J. L., Howell, K. A., Whelan, J., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+analysis+of+the+rice+mitochondrial+proteome.">Towards an analysis of the rice mitochondrial proteome.</a> Plant Physiol. 132 (1), 230-242 (2003).</li></ol>

作者声明没有利益冲突。

通常, 从拟南芥中分离出的线粒体从大约 80-100 3-4 周的老植株中提取出3毫克的线粒体, 尽管在彻底研磨的情况下, 通常能获得大于5毫克的产量。产量随生长条件而变化, 随叶衰老而显著下降, 虽然线粒体结构在衰老过程中似乎保持良好的9。获得良好的产量的最关键的特点之一是磨削溶解细胞释放线粒体的方法。虽然有许多机械研磨装置可供购买, 但在砂浆和杵中的拟南芥研磨在产?...

植物线粒体研究的历史追溯到100年1。完整的线粒体最早是在二十世纪五十年代早期用离心分离法隔离的。二十世纪八十年代, 胶体密度梯度的出现使线粒体得以纯化而不受渗透调节。虽然梯度纯化的线粒体适用于大多数目的, 由于质谱的敏感性, 甚至相对较小的污染物, 可以检测, 并可能被不适当地分配的线粒体位置2。使用自由流动电泳可以去除 plastidic 和过氧化物污染的3, 但自由流电泳是一种高度专门化的技术, 而且不是绝大多数研究所必需的。此外, 在确定蛋白质的位置时, 需要记住的是, 蛋白质的双重或多重靶向性在细胞中发生。100以上的双重靶向蛋白质被描述为叶绿体/质和线粒体4, 并且一些针对线粒体和过氧化物的蛋白质也被称为5。此外, 蛋白质在特定刺激下的重新,例如氧化应激, 是细胞生物学中一个新兴的主题,6。因此, 蛋白质的位置需要考虑的背景下的生物学研究, 并使用各种方法来确定和验证位置2。
线粒体通常是从植物组织中分离出来的, 通过均匀化, 在打破打开细胞壁释放线粒体, 而不是破坏线粒体之间需要平衡。传统上, 与马铃薯和花椰菜, 均匀化涉及使用家用搅拌器/榨汁机的设备, 使液体提取物在缓冲区与各种组成部分, 以维持活动。从豌豆叶分离线粒体 (一种流行的用于线粒体分离的材料, 使用幼苗 (〜10天), 利用搅拌器来溶解细胞, 因为叶子材料是软的。随着拟南芥 T-DNA 插入淘汰线的可用性, 需要能够净化线粒体进行功能研究, 有必要开发方法分离线粒体从叶, 根或花组织.整体上为其他植物开发的方法运作良好7, 与特权, 磨削的材料需要优化。对于拟南芥, 可以在不同的方式 (见下文), 并不同的组织类型 (根与芽)。连续梯度的使用也可以优化, 因为线粒体的密度从不同的器官或发育阶段意味着它们可以迁移不同。因此, 为最大限度的分离密度的梯度可以细化, 以确保达到最佳的分离。
一旦纯化了线粒体可以用于各种研究, 包括蛋白质和 tRNA 摄取实验, 酶活性测定, 呼吸链测量和西方印迹分析。分离的线粒体也可用于质谱分析蛋白质丰度。有针对性的多反应监测 (MRM) 分析允许量化的定义蛋白质, 但需要显着的发展。相比之下, 通过二甲基或其他同位素标记8进行量化, 提供了一种发现方法, 用于识别整个蛋白质组中的差异, 可以用来揭示新的生物学见解。

<table><tbody><tr><td>ADP</td><td>Sigma-Aldrich</td><td>A2754</td><td>Chemical</td></tr><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>AOX antibody</td><td>from Tom Elthon</td><td></td><td>Elthon et al., 1989</td></tr><tr><td>Ascorbate</td><td>Sigma-Aldrich</td><td>A0157</td><td>Ascorbate Oxidase from Cucurbita sp.</td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A26209</td><td>Chemical</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Bovogen</td><td>BSAS 1.0</td><td>Chemical</td></tr><tr><td>Clarity western ECL substrate</td><td>Bio-Rad Laboratories</td><td>1705061</td><td>Chemical</td></tr><tr><td>Criterion Stain-Free Precast Gels 8-16% 18 Wells</td><td>Bio-Rad Laboratories</td><td>5678104</td><td>Chemical</td></tr><tr><td>Cyanide</td><td>Sigma-Aldrich</td><td>60178</td><td>Chemical</td></tr><tr><td>Cytochrome c</td><td>Sigma-Aldrich</td><td>C3131</td><td>Chemical</td></tr><tr><td>Difco Agar, granulated</td><td>BD Biosciences</td><td>214530</td><td>Chemical</td></tr><tr><td>Dithiotreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td>Chemical</td></tr><tr><td>EDTA disodium salt</td><td>Sigma-Aldrich</td><td>E5134</td><td>Chemical</td></tr><tr><td>Gamborg B-5 Basal Medium</td><td>Austratec</td><td>G398-100L</td><td>Chemical</td></tr><tr><td>Gamborg Vitamin Solution (1000x)</td><td>Austratec</td><td>G219-100ML</td><td>Chemical</td></tr><tr><td>Goat Anti-Mouse IgG (H + L)-HRP Conjugate</td><td>Bio-Rad Laboratories</td><td>1706516-2ml</td><td>Chemical</td></tr><tr><td>Goat Anti-Rabbit IgG (H + L)-HRP Conjugate</td><td>Bio-Rad Laboratories</td><td>1706515-2ml</td><td>Chemical</td></tr><tr><td>L-Cysteine</td><td>Sigma</td><td>C7352-100G</td><td>Chemical</td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>230391</td><td>Chemical</td></tr><tr><td>Murashige &amp;amp; Skoog Basal Salt Mixture (MS)</td><td>Austratec</td><td>M524-100L</td><td>Chemical</td></tr><tr><td>Myxothiazol</td><td>Sigma-Aldrich</td><td>T5580</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>NADH</td><td>Sigma-Aldrich</td><td>N8129</td><td>Chemical</td></tr><tr><td>Ndufs4 antibody</td><td>from Etienne Meyer</td><td></td><td>Meyer et al., 2009</td></tr><tr><td>n-Propyl gallate</td><td>Sigma-Aldrich</td><td>P3130</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td>Chemical, colloidal density gradient</td></tr><tr><td>Polyvinylpyrrolidone (PVP40)</td><td>Sigma-Aldrich</td><td>PVP40</td><td>Chemical</td></tr><tr><td>Potassium cyanide</td><td>Sigma-Aldrich</td><td>60178</td><td>Chemical</td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>Sigma-Aldrich</td><td>P5655</td><td>Chemical</td></tr><tr><td>Pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td>Chemical</td></tr><tr><td>Sodium chloride</td><td>Chem-Supply</td><td>SA046</td><td>Chemical</td></tr><tr><td>Sodium dithionite</td><td>Sigma-Aldrich</td><td>157953</td><td>Chemical</td></tr><tr><td>Sodium L-ascorbate</td><td>Sigma</td><td>A4034-100G</td><td>Chemical</td></tr><tr><td>Succinate</td><td>Sigma-Aldrich</td><td>S2378</td><td>Chemical</td></tr><tr><td>Sucrose</td><td>Chem-Supply</td><td>SA030</td><td>Chemical</td></tr><tr><td>TES</td><td>Sigma-Aldrich</td><td>T1375</td><td>Chemical</td></tr><tr><td>Tetrasodium pyrophosphate (Na4P2O7 &amp;middot; 10H2O)</td><td>Sigma-Aldrich</td><td>221368</td><td>Chemical</td></tr><tr><td>Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit</td><td>Bio-Rad Laboratories</td><td>1704271</td><td>Chemical</td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>X100</td><td>Chemical, detergent</td></tr><tr><td>Western Blocking Reagent</td><td>Sigma</td><td>11921681001</td><td>Chemical</td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>XS204</td><td>Equipment</td></tr><tr><td>Beakers</td><td>Isolab</td><td>50 mL</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Avanti J-26XP</td><td>Equipment</td></tr><tr><td>Centrifuge tubes</td><td>Nalgene</td><td>3117-9500</td><td>Equipment</td></tr><tr><td>Circulator</td><td>Julabo</td><td>1124971</td><td>Attached to oxygen electrode chamber</td></tr><tr><td>Conical flask</td><td>Isolab</td><td>500 mL</td><td></td></tr><tr><td>Dropper</td><td></td><td>3 mL</td><td></td></tr><tr><td>Fixed angle rotor</td><td>Beckman Coulter</td><td>JA25.5</td><td>Equipment</td></tr><tr><td>Funnel</td><td>Per Alimenti</td><td>14 cm</td><td>For filtering</td></tr><tr><td>Gradient pourer</td><td>Bio-Rad</td><td>165-4120</td><td>For preparation of gradients</td></tr><tr><td>Magnetic Stirrer ATE</td><td>VELP Scientifica</td><td>F20300165</td><td>Equipment</td></tr><tr><td>Miracloth</td><td>VWR</td><td>EM475855-1R</td><td>Filtration material</td></tr><tr><td>Mortar and pestle</td><td>Jamie Oliver</td><td>Granite, 6 Inch</td><td>Equipment</td></tr><tr><td>O2view</td><td>Hansatech Instruments</td><td></td><td>Oxygen monitoring software</td></tr><tr><td>Oxygraph Plus System</td><td>Hansatech Instruments</td><td>1187253</td><td>Clark-type oxygen electrode</td></tr><tr><td>Paintbrush</td><td>Artist first choice</td><td>1008R-12</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td>plastic paraffin film</td></tr><tr><td>Peristaltic pump</td><td>Gilson</td><td>F155001</td><td>For preparation of gradients</td></tr><tr><td>PVC peristaltic tubing</td><td>Gilson</td><td>F117930</td><td>For preparation of gradients</td></tr><tr><td>Water bath</td><td>VELP Scientifica</td><td>OCB</td><td>Equipment</td></tr></tbody></table>

isolation and respiratory measurements of mitochondria from arabidopsis thaliana

线粒体是植物中许多代谢途径所必需的细胞器, 最显著的是从还原化合物 (如烟酰胺腺嘌呤核苷酸 (NADH) 和黄素腺嘌呤的氧化中生成腺苷三磷酸 (ATP)核苷酸 (FADH2)。完整的注释的拟南芥基因组已经建立了它作为最广泛使用的植物模型系统, 因此需要净化线粒体的各种器官 (叶, 根, 或花) 是必要的充分利用的工具,现在可用于拟南芥研究线粒体生物学。线粒体通过不同的方法与组织的均匀性分离, 接着是一系列的不同的离心步骤, 产生了一种用连续的胶体密度梯度进一步纯化的粗线粒体颗粒。离心.胶体密度材料随后被多个离心步骤去除。从100克的新鲜叶组织开始, 可以常规获得 2-3 毫克的线粒体。呼吸实验在这些线粒体显示典型率 100-250 nmol O2 min-1镁总线粒体蛋白质-1 (NADH 依赖性率) 以使用各种各样的基体和抑制剂的能力确定哪些基板被氧化, 以及替代和细胞色素终端的容量氧化。该协议描述了一种分离方法的线粒体从拟南芥叶片使用连续胶体密度梯度和有效的呼吸测量纯化植物线粒体。

利用该协议, 我们可以通过 SDS 页和免疫检测不同的线粒体蛋白。如图 3A所示, 从水培养组织中分离出来的蛋白质足以检测微弱的带 (2 µg)。信号强度随负载量的增加而增大。对于从板上生长的组织中分离出的线粒体 (图 3B), 可以用免疫替代氧化酶抗体对不同基因型的高光应力处理进行反应。
<p class="jove_content" fo:keep-together.withi...

Watch this Scientific Journal Video about Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana at JoVE.com

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

由于线粒体只占植物细胞的一小部分, 它们需要被纯化以进行一系列的研究。线粒体可以通过同质化的各种植物器官分离, 其次是微分和密度梯度离心, 以获得高纯度的线粒体分数。

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

isolation-respiratory-measurements-mitochondria-from-arabidopsis

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Unit 1

Mitochondria and Energy Production

SCIENTISTS IN ACTION

15.2K 次观看.  La Trobe University. 由于线粒体只占植物细胞的一小部分, 它们需要被纯化以进行一系列的研究。线粒体可以通过同质化的各种植物器官分离, 其次是微分和密度梯度离心, 以获得高纯度的线粒体分数。

视频: Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

科研

发育生物学

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

生物学

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 ...

生物化学

<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 ...

<meta charset="utf8"></head><body>改变线粒体功能以用于治疗和生物杀虫剂应用

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

<meta charset="utf8"></head><body>揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

拟南芥线粒体的分离和呼吸测量

摘要

探索更多视频

此视频中的章节

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

骨骼肌的线粒体分离

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

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

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解