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>

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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量方面取得了一致的良好效果, 因为它裂解细胞器的小损伤。虽然机械磨床是快速的, 他们需要优化, 所需的研磨量可以根据组织的不同。用砂浆和杵, 它往往方便, 更有效的, 如果组织是切片或切割前刀或剪刀磨。如上所述, 所有步骤都需要在4° c 下进行, 从研磨结束到获得被净化的线粒体颗粒的整个过程应该需要大约4小时。由于洗涤剂或其它试剂在管子或梯度 pourers 上的痕迹可以显著降低产量, 所以在这些程序中使用的所有部件都没有洗涤剂洗涤, 也没有在其他程序中使用。最后, 重要的是, 线粒体是足够的分离, 从其他分数的梯度, 让他们被删除和洗涤。如果他们是太接近底部的管, 这意味着混合的轻和重的解决方案需要调整, 让更多的重溶液倒入前混合。反之, 如果波段出现漫反射, 并在管中高, 混合需要发生在稍早。
这里概述的方法可以很容易地用来隔离的线粒体从拟南芥花和根组织11,12。对根系来说, 在水培培养中生长方便, 需要100克的新鲜体重, 获得2毫克的线粒体。在研磨前, 应先将磨根切成小块, 然后在砂浆和杵中或在搅拌机中磨根组织, 从而提高产量。对于线粒体功能通常增强的花组织13, 用砂浆和杵研磨效果很好, 但材料数量有限意味着需要种植大量的植物来收获组织。线粒体已从各种拟南芥器官分离使用类似的方法或轻微的修改14,15 , 从大米 (水稻,)16。
上述方法的局限性是 i) 线粒体隔离所需的大量种子, ii) 仅在本隔离方法中应用的拟南芥和大米的特定组织, iii) 少量的各种污染物 (如体蛋白) 仍然存在于纯化的线粒体中。利用上述方法获得的线粒体适用于各种研究, 包括从吸氧研究到定量质谱分析蛋白质丰度。梯度纯化的线粒体仍将含有少量的各种污染物, 如体蛋白3。与定义的细胞表的比较可以用来确定线粒体蛋白质的变化, 只要 non-mitochondrial 的蛋白质的污染量是小 (< 10%) 和相似的样品比较, 所以类型和程度non-mitochondrial 蛋白是相似的。通过 SDS 页或其他凝方法对蛋白质丰度的分析可以用相对较少的线粒体 (2-20 µg) 进行, 利用不同数量的线粒体检测抗体的线性度 (图 3)。虽然孔 (电压依赖性阴离子通道 (VDAC)) 经常被用作量化的加载控制, 但在我们的经验中, 这种蛋白质的相对较大的丰度可能意味着, 如果大量的蛋白质被装在凝胶上, 反应往往不是线性的。, 因此在使用这种加载控制时, 应始终检查抗体反应的线性度。这种分离线粒体的方法的意义在于, 与使用蔗糖作为材料形成密度梯度的梯度不同, 胶体密度梯度不要求纯化的线粒体渗透调整蔗糖.这意味着, 有较少的机会破裂的纯化线粒体和洗涤后, 以消除胶体密度材料, 它们可以直接用于各种化验或应用。
组织印迹, 从整个叶或组织提取物中检测到线粒体蛋白, 是测量该组织中线粒体蛋白质数量的诱人方法。鉴于线粒体的体积与其他细胞器相比, 线粒体蛋白在整个组织提取物中的检测需要谨慎地解释, 因为线粒体蛋白在这种方法中可能无法被检测到。仔细的控制, 在那里纯化的线粒体电泳连同组织提取物, 以确保相同的迁移和线性检测, 需要进行对这种方法的信心。
在克拉克型氧电极的帮助下, 可以测量溶液中氧分压的变化。实际电极包括铂阴极和银阳极, 由氯化钾桥连接, 由电解质润湿的纸 (卷烟纸) 和氧气渗透膜 (聚四氟乙烯膜) 覆盖。电压为 600-700 mV 导致氧气的减少, 给氧浓度和电压之间的线性关系。氧电极可从不同的公司获得。每个公司将有自己的指示, 关于组装和设置氧电极。然而, 在一般情况下, 电极盘的银阳极和铂阴极需要用电极清洗套件或橡皮擦笔在装配前进行清洗。重要的是要确保在组装过程中气泡不形成 (例如, 在聚四氟乙烯隔膜和卷烟纸之间的)。组装后, 电极盘需要附着在反应室基底上的铂阴极向上的腔室。会议厅本身是由一个水夹克, 确保温度控制包围。这是非常重要的是, 该会议厅总是留满水, 因为膜将干燥和裂缝, 否则。同样, 在将化合物添加到腔室时, 管或注射器不得触及膜, 以避免撕裂。
在化验前, 呼吸介质应加热到与试验室相同的温度 (在大多数情况下为25° c), 并且应允许膜在呼吸缓冲中平衡几分钟。在关闭柱塞进行氧耗测定后, 应使用一次性升注射器 (例如微型注射器) 或一次性的凝胶加载吸管贴士来进行效应分子的添加。如果使用微型注射器, 必须确保注射器是彻底冲洗与100% 乙醇和水之间的补充, 以避免污染的库存解决方案。这在测量之间也申请房间的洗涤。大多数用于氧耗测定的试剂都可溶于水, 并且可以很容易地通过多次冲洗 (大约五次) 在测量之间去除。然而, 一些用于化验的化学物质只能溶于有机溶剂, 如 antimycin、噻唑和 nPG。因此, 房间需要用有机溶剂 (50% (v/v) 乙醇) 冲洗, 以去除这些分子的残余痕迹, 然后用水 (大约五倍) 来耗尽残渣。

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 &#38; 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 &#183; 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。
线粒体的完整性, 细胞色素c通路活动和替代通路可以用新近分离的线粒体样本来测量。在描述的隔离过程 (图 4A) 中, 线粒体完整性得到了很好的保存。添加不同的基质, 抑制剂和影响到孤立的线粒体, 氧气消耗通过细胞色素c通路和替代氧化酶通路 (图 4B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56627/56627fig1.jpg"> 图 1: 渐变准备的设置.左: 离心管 (50 毫升) 与一个轻微的角度放置在冰上的 PVC 蠕动油管插座贴在管内;中间: 蠕动泵;右: 梯度倒在含有重梯度 (内腔) 和光梯度 (外腔) 溶液的磁力搅拌器的顶部。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56627/56627fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56627/56627fig2.jpg"> 图 2: 使用连续 0-4.4% (w/v) PVP/28%colloidal 密度梯度对拟南芥芽中的线粒体进行纯化.暗绿色带是类和其他污染物, 如在梯度溶液顶部所示。线粒体的分数呈白色带绿色的带子, 靠近管子的底部。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56627/56627fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56627/56627fig3.jpg"> 图 3: 使用特定抗体的 SDS 页和免疫分离线粒体蛋白.(A) 从两周前的 water-cultured拟南芥野生型 (Columbia-0、Col-0) 植物中分离出的线粒体被置于 SDS 页上, 并对 NADH 的抗体进行了探测: 泛氧化亚单位 S4 (Ndufs4, At5g67590)。将分子量不标记物装在凝胶的外车道上, 以公斤道尔顿 (kDa) 表示十代表带的大小。检测到的 Ndufs4 蛋白的表观分子质量为 18 kDa。蛋白质丰度显示相对于2µg 总蛋白的价值。(B) 在 Gamborg 的 B5 培养基上生长的两周老苗, 3% (w/五) 蔗糖和0.8% 琼脂 (w/v) 被暴露到750µE m-2 s-1高亮 (HL), 并在 6 h 之后收获. 线粒体被纯化, 线粒体蛋白用 SDS 页分离, 并对替代氧化酶 (AOX) 的抗体进行了探讨。immunodetectable AOX 蛋白的存在与表面的分子质量 34 kDa 被表明。蛋白质丰度显示相对于控制值 (2 µg)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56627/56627fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56627/56627fig4.jpg"> 图 4: 孤立植物线粒体对 O2消耗量的代表性跟踪.(A) 从拟南芥野生型植物 (Col-0) 中分离出的线粒体在耗氧量测量前对外膜完整性进行了分析。30µL 分离的线粒体 (150 µg 总线粒体蛋白) 被使用。线粒体完整性被确定为90% 如上所述。(B) 使用30µL 分离的线粒体 (150 µg 总线粒体蛋白) 从拟南芥野生型植物中进行耗氧量测量。测定线粒体总呼吸和 AOX 通路。通过这些数据, 可以确定通过细胞色素c通路和 AOX 通路的耗氧量。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56627/56627fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">研磨介质</td>
		</tr>
<tr>
<td>化学</td>
			<td>浓度</td>
		</tr>
<tr>
<td>蔗糖</td>
			<td>0.3 M</td>
		</tr>
<tr>
<td>钠焦磷酸 (Na4P2O7 · 10 H2O)</td>
			<td>25毫米</td>
		</tr>
<tr>
<td>EDTA 二钠盐</td>
			<td>2毫米</td>
		</tr>
<tr>
<td>磷酸二钾 (KH2PO4)</td>
			<td>10毫米</td>
		</tr>
<tr>
<td>酮 (PVP40)</td>
			<td>1% (w/五)</td>
		</tr>
<tr>
<td>牛血清白蛋白 (BSA)</td>
			<td>1% (w/五)</td>
		</tr>
<tr>
<td>去离子水</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">将 pH 值调整到 7.5 (使用 HCl)</td>
		</tr>
<tr>
<td colspan="2">注: 对于300毫升研磨介质, 1.06 克钠抗坏血酸 (最终浓度: 17.84 mm) 和0.74 克半胱氨酸 (最终浓度: 20.36 mm) 在使用前添加。检查 pH 值后添加和调整到7.5 与1米氢氧化钠, 如果需要。</td>
		</tr>
<tr>
<td colspan="2">2X 洗涤缓冲器</td>
		</tr>
<tr>
<td>化学</td>
			<td>浓度</td>
		</tr>
<tr>
<td>蔗糖</td>
			<td>0.6 M</td>
		</tr>
<tr>
<td>附加</td>
			<td>20毫米</td>
		</tr>
<tr>
<td>牛血清白蛋白 (BSA)</td>
			<td>0.2% (w/五)</td>
		</tr>
<tr>
<td>去离子水</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">调整 pH 值至 7.5 (使用氢氧化钠)</td>
		</tr>
<tr>
<td colspan="2">梯度</td>
		</tr>
<tr>
<td>重梯度溶液 (4.4% (w/v) PVP)</td>
			<td>2渐变管</td>
		</tr>
<tr>
<td>2X 洗涤缓冲器</td>
			<td>17.5 毫升</td>
		</tr>
<tr>
<td>Colloidial 密度梯度</td>
			<td>9.8 毫升</td>
		</tr>
<tr>
<td>PVP-40 (20% (w/五))</td>
			<td>7.7 毫升</td>
		</tr>
<tr>
<td>光梯度溶液 (0% (w/v) PVP)</td>
			<td>2渐变管</td>
		</tr>
<tr>
<td>2X 洗涤缓冲器</td>
			<td>17.5 毫升</td>
		</tr>
<tr>
<td>Colloidial 密度梯度</td>
			<td>9.8 毫升</td>
		</tr>
<tr>
<td>去离子水</td>
			<td>7.7 毫升</td>
		</tr>
</tbody></table>表 1: 用于线粒体隔离的缓冲器和渐变的组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>缩写</td>
			<td>库存解决方案的集中</td>
			<td>存储</td>
			<td>最终浓度</td>
			<td>增加1毫升反应的体积</td>
		</tr>
<tr>
<td colspan="6">基</td>
		</tr>
<tr>
<td>细胞色素 c</td>
			<td>Cyt c</td>
			<td>2.5 mM (在 H2O)</td>
			<td>-20 ° c</td>
			<td>25μ m</td>
			<td>10μ l</td>
		</tr>
<tr>
<td>nadh</td>
			<td>nadh</td>
			<td>0.1 M (在 H2O)</td>
			<td>-20 ° c</td>
			<td>1毫米</td>
			<td>10μ l</td>
		</tr>
<tr>
<td>琥珀酸</td>
			<td>succ</td>
			<td>500 mM (在 H2O)</td>
			<td>-20 ° c</td>
			<td>5毫米</td>
			<td>10μ l</td>
		</tr>
<tr>
<td colspan="6">抑制剂</td>
		</tr>
<tr>
<td>Antimycin</td>
			<td>aa</td>
			<td>1毫米 (在乙醇)</td>
			<td>-20 ° c</td>
			<td>5μ m</td>
			<td>5μ l</td>
		</tr>
<tr>
<td>氰</td>
			<td>kcn</td>
			<td>100 mM (在 H2O)</td>
			<td>4° c</td>
			<td>1毫米</td>
			<td>10μ l</td>
		</tr>
<tr>
<td>噻唑</td>
			<td>Myxo</td>
			<td>500μ m (在乙醇)</td>
			<td>-20 ° c</td>
			<td>2.5 μ m</td>
			<td>5μ l</td>
		</tr>
<tr>
<td>
n-没食子酸丙酯</td>
			<td>npg</td>
			<td>100毫米 (在乙醇)</td>
			<td>-20 ° c</td>
			<td>500μ m</td>
			<td>5μ l</td>
		</tr>
<tr>
<td colspan="6">效应</td>
		</tr>
<tr>
<td>adp</td>
			<td>adp</td>
			<td>100 mM (在 H2O)</td>
			<td>-20 ° c</td>
			<td>1毫米</td>
			<td>10μ l</td>
		</tr>
<tr>
<td>抗坏血酸</td>
			<td>asc</td>
			<td>500 mM (在 H2O)</td>
			<td>在使用的那天做新鲜的</td>
			<td>10毫米</td>
			<td>20μ l</td>
		</tr>
<tr>
<td>atp</td>
			<td>atp</td>
			<td>100 mM (在 H2O)</td>
			<td>-20 ° c</td>
			<td>500μ m</td>
			<td>5μ l</td>
		</tr>
<tr>
<td>Dithiotreitol</td>
			<td>dtt</td>
			<td>1 M (在 H2O)</td>
			<td>在使用的那天做新鲜的</td>
			<td>10毫米</td>
			<td>10μ l</td>
		</tr>
<tr>
<td>丙酮</td>
			<td>pyr</td>
			<td>1 M (在 H2O)</td>
			<td>-20 ° c</td>
			<td>10毫米</td>
			<td>10μ l</td>
		</tr>
<tr>
<td>洗涤剂</td>
			<td></td>
			<td>10% (v/v) (在 H2O)</td>
			<td>4° c</td>
			<td>0.05% (v/五)</td>
			<td>5μ l</td>
		</tr>
</tbody></table>表 2: 用于耗氧量测量的基底、抑制剂和效应物清单。

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

Nanyang Technological University

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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

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

科研

生物化学

Rapid Isolation of the Mitoribosome from HEK Cells

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

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

HEK 细胞 Mitoribosome 的快速分离

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

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

寄生性锥虫锥虫锥虫的 f1-atpase 的分离

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

拟南芥叶绿体子隔层的制备及免疫印迹蛋白质组化分析

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

组织细胞外囊泡与肝脏分离

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

呼吸系统同步病毒病毒特定核胶囊的生成和组装

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

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

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

骨骼肌线粒体蛋白质导入能力的测定

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