我们感谢 IGBMC 高吞吐量测序设施的所有工作人员, 即 "法国 Génomique" 财团 (ANR10-INBS-09-08) 的成员和所有 IGBMC 一般事务人员, 特别是 IGBMC 动物设施的工作人员。这项工作得到了来自 CNRS、INSERM、AFM、甲国家组织勒癌症的赠款、法国全国基金在 Investissements 艾文莉标记 ANR-10-IDEX-0002-02、Labex INRT ANR-10-IDEX-0001-02 和ANR-AR2GR-16-CE11-009-01资助者在研究设计、数据收集和分析、决定出版或准备原稿方面没有作用。甲组织的支持下, 美国的 ANR-AR2GR-16-CE11-009-01, 和五, U 由 Ministère de Enseignement 等研究。ID 是一个 ' équipe labellisée ' 的甲国立组织勒癌症。

根据国家动物保育指导方针 (欧洲委员会指令 86/609/中欧和东欧), 按照有关实验室动物的护理和使用的机构准则, 对老鼠进行了保存;法国法令 87-848)。所有程序均由法国国家伦理委员会批准. 
 1. 肌肉组织的分离 <ol> <li> 由颈椎脱位牺牲一成人6至8周大鼠。Sterlise 用70% 乙醇冲洗四肢, 用细点剪刀和镊子解剖后肢肌肉 (腓肠肌、胫骨前、四头肌). 
	注意: 一只老鼠应该产生大约500毫克的组织. </li>
	<li> 将2毫升测试管中的肌肉剁碎, 其中含有1毫升的 ice-cold 低渗缓冲液, 以均匀制备小 (#60; 2-3 mm 3 ) 件使用细剪刀和离开管晃动在一个板凳顶部搅拌器在4和 #176; C 为 5-10 min. </li>
</ol> 2。组织裂解 注意: 对于所有的缓冲区组合, 请参阅 表 1 。
<ol> <li> 将匀浆转移到14毫升的圆底管和重5毫升的冷低渗缓冲液 (无 EDTA 蛋白酶抑制剂鸡尾酒和 PMSF). 
	<ol> <li> 使用松散的 dounce (3 分钟内 20-30 冲程) 或机械组织均质体在 15-三十年代的圆底14毫升管中必须碎肌肉组织. 
		注: 在这个阶段, 可以通过光显微镜对裂解的效率进行评估, 如果需要, 还可以进行额外的干扰. </li>
	</ol> </li> <li> 使用冷低渗缓冲将匀浆转移到15毫升管和填充体积到10毫升。在继续下一只鼠标之前, 按照下面的描述修复匀浆. </li>
</ol> 3。固定 <ol> <li> 将甲醛添加到1% 的最终浓度, 并在室温下摇动10分钟. </li>
	<li> 在每管中加入甘氨酸最终浓度为0.125 米, 以停止固定和摇动 #160; 5-10 分钟室温. </li>
</ol> 4。核准备工作 注意: 请参阅 表 1 中的所有缓冲区组合。
<ol> <li> 使用松散的 dounce (5-10 笔画) 必须固定的裂解产物. 
	<ol> <li> 传输到15毫升管和离心机在 1000 x g 5 分钟, 在4和 #176; C 获得细胞核和细胞碎片. </li>
	</ol> </li> <li> 在新鲜的5毫升低渗缓冲器中移除上清和重颗粒。通过70和 #181 将裂解液过滤到50毫升的管中。重新过滤滤液通过40和 #181; m 细胞过滤器. </li>
	<li> 将滤液转移到15毫升管和离心机在 1000 x g 5 分钟, 在4和 #176; C 以获得核颗粒. 
	注: 在这个阶段, 原子核可以立即声, 或在液氮中以干球的形式被冻结, 并储存在-80 和 #176; C. </li>
</ol> 5。超声 <ol> <li> 评估核颗粒的体积 (大约50和 #181; L 为机械和 dounce 化) 和重它在超声缓冲3到4倍包装的核容量和几种为 10-15 min 在4和 #176; C 使用 sonicator. 
	注: 染色质可按以下描述立即分析 (6) 或冷冻保存在-80 和 #176; C. </li>
</ol> 6。评估超声的染色质碎片大小 注意: 请参阅 表 1 中的所有缓冲区组合。
<ol> <li> de-crosslink, 取30和 #181; 1.5 毫升试管中的染色质, 并添加20和 #181; l 5 M 氯化钠。完成卷到500和 #181; L 和孵育在65和 #176; C 过夜. 
	<ol> <li> 第二天, 使用1和 #181 进行处理; l 蛋白酶 K (20 毫克/毫升), 10 和 #181; l 2 米, pH 6.8, 10 和 #181; 0.5 米 EDTA 为1小时, 在42和 #176; c. 执行苯酚-氯仿/氯仿萃取, 并沉淀的 DNA 1 量的 acetat 钠e (3 M) 和2容量100% 乙醇为一 h 在-80 和 #176; c 或隔夜在-20 和 #176; c. </li>
	</ol> </li> <li> 通过离心在 13500 x g 上进行15分钟的醒酒, 用冷70% 乙醇将颗粒彻底洗净, 再离心5分钟醒酒上清液并风干颗粒. </li>
	<li> 重原始卷 (30 和 #181; L) 中的颗粒。在 260 nm/280 纳米的光度法测定分光光度计中的 DNA 浓度。吸管 500-1000 ng dna 与 dna 大小梯在单独的车道上的1.5% 琼脂糖凝胶和执行电泳评估的片段大小染色质. 
	注意: 碎片大小应介于 200-500 碱基对之间, 并且不应有可检测到的高分子量片段对应于或不完整的 DNA。如果需要, 染色质溶液 (步骤 5) 可以 re-sonicated 较长时间, 然后重新如上所述, 直到获得最佳尺寸. </li>
</ol> 7。染色质沉淀 (芯片) 注意: 请参阅 表 1 中的所有缓冲区组合。
<ol> <li> 在乙醇中通过 aliquoting 1 毫升的50% 浆块将蛋白 G 琼脂珠阻止。在台式离心机中离心 400 x g, 用1毫升的 TE 缓冲离心机在 400 x 克上进行颗粒化, 然后重复洗涤. 
	<ol> <li> 在第二次离心后, re-suspend 在1毫升的芯片稀释缓冲器中, 25 和 #181; BSA (库存20毫克/毫升) 和20和 #181; l 酵母 tRNA (库存10毫克/毫升)。在4和 #176 中, 用旋转 (40-60 rpm) 将珠子至少阻挡在2小时前; C. </li>
		<li> 稀释50和 #181; g 的染色质 (步骤 5) 8 至10次使用芯片稀释缓冲器。添加50和 #181; 在4和 #176 中, 在2° c 旋转 (40-60 rpm) 上, 将被阻挡的珠浆和孵育的 L. 在400和 #176 上离心, 在 4 x g 处获得名册染色质并将其转移到新鲜的试管中. </li>
	</ol> </li> <li> 对于沉淀, 添加适当数量的主要抗体 (, 如 , 1-2 和 #181; g 每10和 #181; g), 并在夜间孵育4和 #176 的旋转; C. 
	注: 最佳抗体量可以由供应商推荐, 也可以通过执行不同数量的抗体的 qPCR 来确定, 直到最佳的富集被观察到。此外, 蛋白质 G 琼脂可能被蛋白质琼脂所取代, 这取决于所使用抗体的亚型. </li>
	<li> 添加被阻止的珠子第二天和孵化1小时与旋转 (40-60 rpm) 在4和 #176; c. 离心机在 400 x g 为 20-三十年代, 以颗粒的珠子, 清除上清, 并开始切屑洗. </li>
	<li> 芯片洗涤: 执行以下洗涤与缓冲: 一次以低盐缓冲, 然后两次与高盐缓冲, 两次与氯化缓冲, 和最后两次与 TE 缓冲. 
	<ol> <li> 执行每洗10分钟的旋转 (40-60 rpm), 在4和 #176; C 和颗粒的珠之间的每个洗涤的离心在 400 x g 为 20-三十年代在一个台式离心机. </li>
	</ol> </li> <li> 洗脱时, 删除最后的洗涤和重250和 #181 中的珠子; L 洗脱缓冲液 (新鲜准备) 在室温下15分钟, 在晃动时. </li>
	<li> 离心在 400 x g 和收集洗在一个新鲜的管。执行此步骤两次, 然后 de-crosslink 洗一夜20和 #181; l 氯化钠5米和1和 #181; 核糖核酸 A (10 毫克/毫升), 用蛋白酶 K 和苯酚-氯仿/氯仿机治疗如步骤6所示. </li>
	<li> 重在50和 #181 中, 根据标准的协议使用等分进行芯片 qPCR 15 检查芯片的质量. </li>
</ol>

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作者声明他们没有竞争的金融利益。

在这里, 我们描述了一个新的协议, 从成年小鼠骨骼肌肉制备染色质, 并表明, 这种染色质是适用于芯片实验, 检测转录因子结合和共价键组蛋白修饰肌纤维核。此协议涉及几个关键步骤。第一种是组织破坏, 可以通过 dounce 或机械剪切来进行。机械剪切速度快, 重现性好, 因此是选择的方法。不过, 如果没有合适的仪器, 也可以由 douncing, 在进行固定步骤之前, 建议在显微镜下对裂解效率进行评估。固定的全组织裂解, 而不是固定的孤立核, 允许更多的可再生超声在较小的体积和短期内。我们还选择在中断后修复裂解, 而不是在中断之前修复组织, 如托马斯et al.所描述的那样。10预修复组织导致化效率降低。该协议的基础上, 机械剪切也比其他建议的解决方案, 如胶原酶消化, 这涉及长期孵化的解剖组织在37° c8。在此期间, 基因表达和转录因子结合的变化可能发生。因此, 尽可能快地修复细胞核是更忠实地捕捉正常生理状态的关键。
第二个关键步骤是从固定的裂解液中提纯原子核。为此, 顺序过滤固定的裂解液, 首先通过70µm 细胞过滤器, 以消除较大的碎片, 然后通过一个40µm 细胞过滤器, 以消除罚款的碎片, 避免堵塞的滤器和最大的产量的核获得。经过滤纯化后, 细胞核声, de-crosslinking 后确定 DNA 片段大小。一个典型的准备从两个后肢的一只老鼠产生100µg 的染色质。将裂解从多达3只老鼠中收集起来, 可以通过减少制剂中的损失来提高产量。这一过程是有效的, 因为它允许恢复多达75% 的基因组 DNA 染色质 (数据没有显示)。
用于评估转录因子结合和后生修饰的芯片实验证明了该协议对于研究肌纤维细胞核基因调控的适用性。强大的和强健的芯片序列信号 ii 和 H3K27ac 是由染色质产生的, 允许识别肌肉纤维的身份基因由其独特的 H3K27ac 和政客 ii 配置文件7。与卫星或内皮细胞中表达的基因相比, 在纤维核中强烈表达的 H3K27ac 和 II. 类基因的含量更高, 这表明信号主要来自纤维核。这也证实了 Tead4 芯片信号的丢失使用染色质从老鼠那里它是特别地被灭活在肌肉纤维。然而, 对于诸如Vcam1、 Pax7 (未显示的数据) 等卫星单元标记, 较弱但明显高于背景信号的情况, 可以检测出这些细胞的染色质也存在。我们预计, 我们的协议应适用于其他技术, 使用甲醛固定染色质, 如3碳/4 c 和细胞核染色质构象捕获技术。使用我们的协议结合芯片序列的转录因子和染色质修饰与染色质构象捕获应该在未来允许详细了解的基因调控机制的肌肉纤维和如何调节这些可能或扰乱生理刺激, 如运动, 空腹, 高脂肪饮食, 失神经支配, 或在疾病中, 使用染色质制备的基因修饰小鼠复制疾病如 X 连锁中央肌病14。
总之, 这种低成本和时间的协议允许隔离的肌肉纤维核, 可以立即声或冻结在-80 ° c, 为不可告人的使用。使用该协议制备的染色质提供了第一个基因组宽的芯片序列数据从肌肉纤维7 , 并将促进未来的研究, 基因调控在这个组织。

染色质沉淀 (芯片) 结合定量聚合酶链反应 (qPCR) 和高通量测序 (芯片序列) 已成为选择的方法研究转录和表观遗传调控的基因表达在各种组织和单元格类型1。这项技术允许基因组范围内共价染色质修饰, 蛋白的变体占用, 和转录因子绑定2,3。
虽然从培养细胞中执行芯片是很好的建立, 从哺乳动物组织芯片仍然是更具挑战性。为芯片准备染色质涉及几个关键步骤, 需要优化每一个组织和细胞类型。染色质可以从细胞裂解培养细胞或在其细胞核纯化后制备。在哺乳动物组织的情况下, 有效的裂解, 甲醛固定和纯化的细胞核是至关重要的, 以确保最佳的恢复染色质。此外, 选择是否修复核之前或之后, 他们的纯化必须实验确定。尽管有这些障碍, 芯片已成功地从组织, 如肝脏, 睾丸, 或大脑4,5,6。在骨骼肌肉的情况下, 破坏这种身体上的抗性组织, 它表现出高含量的结构蛋白, 和孤立的细胞核是特别具有挑战性的。鉴于这种特异性, 为其他组织优化的协议不会给骨骼肌肉带来令人满意的结果。
在这里, 我们描述了一个协议, 隔离芯片级染色质从小鼠骨骼肌组织, 包括物理扰乱组织, 甲醛固定, 然后分离细胞核和超声。通过对各种转录因子、RNA 聚合酶 II 和共价组蛋白修饰的芯片 qPCR 和芯片序列进行了研究, 证明了该方法制备适合于该组织芯片的染色质的效率7。
这种新的技术比以前报告的协议8要快得多, 包括长时间胶原酶消化步骤, 在此期间, 转录因子基因组本地化的变化和基因表达的改变可能会发生。原子核被隔绝和固定的快速, 使这里描述的方法特别有吸引力准备染色质更加忠实地捕获本机基因组占有状态。此外, 虽然其他方法的染色质分离或蛋白质提取从肌肉组织已被描述8,9,10 , 并用于芯片 qPCR 实验选定的基因, 没有芯片 seq已报告使用这些数据。所报告的方法适用于转录因子和组蛋白修饰芯片序列, 因此也应适用于 3/4 c 或细胞核染色质构象捕获应用。

<table>
	<tbody>
		<tr>
			<td>T 25 digital ULTRA-TURRAX</td>
			<td>T 18 ULTRA-TURRAX</td>
			<td>0009022800</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>SIGMA-ALDRICH CHIMIE SARL</td>
			<td>P7626-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail-EDTA Free</td>
			<td>Roche Diagnostics</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Sepharose beads</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P-3296</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P-2308</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers 70um</td>
			<td>Corning BV</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers 40um</td>
			<td>Corning BV</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde EM grade</td>
			<td>Euromedex</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Fischer Scientific</td>
			<td>12091039</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P3803</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>B4287-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast tRNA</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>R5636</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Blue</td>
			<td>AMIBION</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Pol II ChIP Antibody</td>
			<td>Santa Cruz</td>
			<td>SC-9001</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27ac ChIP Antibody</td>
			<td>Active Motif</td>
			<td>39133</td>
			<td></td>
		</tr>
		<tr>
			<td>Tead4 ChIP Antibody</td>
			<td>Aviva Systems Biology</td>
			<td>(ARP38276_P050)</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>I-3021</td>
			<td></td>
		</tr>
		<tr>
			<td>E220 Focused Ultrasonicator</td>
			<td>Covaris E220</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Additional items</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Loose Dounce</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml and 50 ml falcon tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml round bottom tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml and 2 ml Eppendorf tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m and 40 &#956;m cell strainers (Corning)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

improved protocol for chromatin immunoprecipitation from mouse skeletal muscle

我们描述了一个有效的和可复制的协议, 从成年小鼠骨骼肌的染色质制备, 一个具有高含量结构蛋白的身体抗性组织。从成年小鼠身上解剖肢体肌肉被机械化, 或切碎和 douncing 的组合, 在低渗缓冲液中, 在甲醛固定的细胞裂解。固定核通过机械化或 douncing 的进一步循环纯化, 并连续过滤去除细胞碎片。纯化后的核可以立即声或在冷冻后的后期进行。染色质可以有效地声, 适用于染色质沉淀实验, 如为转录因子、RNA 聚合酶 II 和共价键组蛋白修饰所获得的剖面所示。使用本协议所制备的染色质检测到的绑定事件主要是在肌纤维核中发生的, 尽管其他纤维相关的卫星和内皮细胞存在染色质。因此, 本议定书适用于研究成年小鼠骨骼肌的基因调控。

为了分离细胞核, 我们在15或四十五年代, 在1.8万和 2.2万 rpm 进行了解剖和碎肌组织的机械均质化 (参见材料表)。在所有条件下, 细胞核可以与组织的碎片分离, 但产量是最好的使用较低的速度 (图 1A-b)。原子核也可以由 douncing 为 3-5 min (图 1A, 和数据没有显示), 但机械均匀化是选择的方法, 因为最理想的产量的原子核可以实现在尽可能少的十五年代, 使时间的重要增益, 特别是如果必须处理多个示例 (图 1B)。使用该协议, 多达 2.7 x 107核从500毫克的组织 (相当于1鼠标的组织) 可以隔离, 以产生100µg 染色质。
为了优化染色质碎片从固定核, 我们声5至20分钟。在设置 (请参阅材料表) 下, 10 分钟超声是最佳生成染色质, 平均片段大小为 250 bp (图 2)。在考虑到所使用的 sonicator 类型的情况下, 应该对这一步骤进行优化试验。
为了评估上述协议所制备的肌肉染色质的质量, 对转录因子、组蛋白修饰或 RNA 聚合酶 ii (ii) 进行了芯片 qPCR 和芯片-seq 实验。qPCR 对糖皮质激素受体 (gr) 的存在或缺乏地塞米松 (德克斯) 治疗显示, 强德克依赖性 gr 丰富的调控元素的Redd1和Murf1基因, 已知是德克和 gr调节肌肉纤维11, 而在睾丸特定的Prm1启动子中没有出现信号, 用作负控制 (图 3A)。
qPCR 对乙酰赖氨酸27的组蛋白 H3 (H3K27ac), 在活性增强剂和促进剂中发现的共价修饰, 显示在肌纤维活性的蛋白 (Des) 启动子上的强富集, 与负基因控制区域 (图 3B)。H3K27ac 芯片-seq 在整个Des轨迹中显示了这个标记的高水平, 这是典型的 "super-enhancer" 调节组织识别基因12 (图 4A)。同样地, 在这个轨迹上, 二级芯片也显示出高水平的转录。
qPCR 转录因子 Tead4, 其中发挥重要作用, 在肌细胞分化13,7, 揭示了其丰富的前描述的绑定站点在Ccnd1和Ifrd1基因 (图 3C)。重要的是, 在肌肉纤维 (Tead4skm-)7 (图 3C) 中, 利用小鼠的染色质制备了 Tead4, 从而减少了浓缩。对 Tead1 和 Tead4 的芯片序列数据的分析显示, 它们绑定到Kdm5a基因启动子中的一个站点, 使用来自野生型肌肉的染色质, 而 Tead4 信号, 但不是 Tead1, H3K27ac, 或第二号, 是失去了使用染色质从Tead4skm小鼠的肌肉 (图 4B)。类似地, Tead1 和 Tead4 绑定到Adssl1基因下游的调节元素, 从野生型肌肉中看到染色质, 而 Tead4 绑定则是选择性地从Tead4skm-/肌肉中使用染色质丢失 (图 4C)。这些观察表明, 在芯片 seq 数据中看到的 Tead4 信号来自于肌肉纤维的结合。
为了确定与肌纤维相关的其他细胞类型对芯片 seq 结果的贡献, 我们评估了在肌肉卫星细胞和Pecam1中活跃的Vcam1基因中的 H3K27ac 和 II 信号, 这是一个标记血管内皮细胞能灌溉肌肉。与在Des或Adssl1基因中看到的高水平相比, 在Vcam1和Pecam1基因中看到的 H3K27ac 和 II 型的水平要低得多 (图 4A和图 4C)。与相关组织中表达的肌肉纤维表达基因的信号差异很大, 这表明, 使用上述协议制备的染色质芯片中的信号主要来源于肌肉纤维。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56504/56504fig1.jpg"> 
图 1: 从成年老鼠肌肉中提纯细胞核.(A)由机械化 (1.8万 rpm, 四十五年代) 和 dounce 均匀化 (30 strokes/3 分钟) 所获得的组织裂解, 用台盼蓝染色, 在数字显微镜下观察, 图像在20X 倍放大时被捕获。缩放条 = 100 µm. (B)在所述条件下制备的500毫克组织 (相当于1只老鼠) 获得的细胞核数, 1.8万和 2.2万 rpm 为15和四十五年代或3分钟的 douncing。误差线代表平均± S.E.M. 为 n = 3 只老鼠为机械均匀化, 并且 n = 3 只老鼠为 douncing。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig1large.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/56504/56504fig2.jpg"> 
图 2: 染色质碎片大小的验证.被隔绝的核被声 5, 10, 15 或 20 min 如表明。在 de-crosslinking 和纯化后, 用琼脂糖凝胶电泳对溴溴化物进行了 DNA 片段大小的评估。M 是 DNA 大小标记。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig2large.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/56504/56504fig3.jpg"> 
图 3: 用于芯片 qPCR 的染色质的使用.(A)小鼠受腹腔注射的车辆或地塞米松 (10 毫克/千克) 和1.5 小时后, 染色质是由后肢肌肉制备的。对 GR 靶基因的富集在芯片的Redd1和Murf1,相比, 负控制鱼精蛋白启动子 (Prm1), 表示为输入沉淀的%。(B)芯片用于 H3K27ac 和 IgG 控制抗体, 并在蛋白启动子上进行分析, 并与基因负控制区进行比较。(C) Tead4 芯片是对从野生类型和肌肉特异性 Tead4 击倒小鼠 (Tead4skm-) 获得的肌肉染色质进行的, 并在Tead4和Ccnd1启动子中的 Ifrd1 绑定站点上进行分析, 与控制基因区域。在所有的实验中, 3 只小鼠的染色质被汇集, 误差条代表了三技术复制的学生 T 检验中的平均±东南亚 M。P 值和 #60; 0.001 **, #60; 0.0001 **;NS, 显著。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig3large.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/56504/56504fig4.jpg"> 
图 4: 肌纤维染色质的基因组图谱.(A)大学的截图加利福尼亚州的 y 在圣克鲁斯基因组浏览器的指示位点说明了高得多的 H3K27ac 和第二类蛋白基因, 高表达的肌肉纤维, 与Vcam1和Pecam1基因表达的卫星细胞和内皮细胞, 分别。(B) UCSC 的屏幕截图, 表明 Tead1 和 Tead4 的结合, 以及 H3K27ac 和油料 II, 在染色质从野生型 (重量) 肌肉相比, 从 Tead4skm/- (MT) 肌肉, 选择性失去 Tead4 结合是观察.在 GEO 数据库中, 可作为 GSE82193 提供芯片 seq 数据。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig4large.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>缓冲区名称</td>
			<td colspan="3">组成</td>
		</tr>
		<tr>
			<td rowspan="6">低渗缓冲器</td>
			<td colspan="3">10毫米 HEPES-KOH (pH 7.3)</td>
		</tr>
		<tr>
			<td colspan="3">10毫米氯化钾</td>
		</tr>
		<tr>
			<td colspan="3">5 mM 氯化镁2</td>
		</tr>
		<tr>
			<td colspan="3">0.1% NP-40</td>
		</tr>
		<tr>
			<td colspan="3">0.1 毫米 PMSF (新鲜地增加)</td>
		</tr>
		<tr>
			<td colspan="3">蛋白酶抑制剂鸡尾酒 (PIC) 1x (新鲜添加)</td>
		</tr>
		<tr>
			<td rowspan="8">超声缓冲区</td>
			<td colspan="3">1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">10毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20毫米三盐酸盐 pH 值8</td>
		</tr>
		<tr>
			<td colspan="3">150毫米氯化钠</td>
		</tr>
		<tr>
			<td colspan="3">0.1% 胆酸钠</td>
		</tr>
		<tr>
			<td colspan="3">1% 海卫 X-100</td>
		</tr>
		<tr>
			<td colspan="3">新鲜增加的0.1 毫米 PMSF</td>
		</tr>
		<tr>
			<td colspan="3">PIC 1x</td>
		</tr>
		<tr>
			<td rowspan="7">芯片稀释缓冲器</td>
			<td colspan="3">0.01% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1.1% 海卫 X-100</td>
		</tr>
		<tr>
			<td colspan="3">1.2 毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">16.7 毫米三盐酸 pH8</td>
		</tr>
		<tr>
			<td colspan="3">167毫米氯化钠</td>
		</tr>
		<tr>
			<td colspan="3">新鲜增加的0.1 毫米 PMSF</td>
		</tr>
		<tr>
			<td colspan="3">PIC 1x</td>
		</tr>
		<tr>
			<td rowspan="5">低盐缓冲器</td>
			<td colspan="3">0.1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1% 海卫 X-100</td>
		</tr>
		<tr>
			<td colspan="3">500毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20毫米三盐酸 pH8</td>
		</tr>
		<tr>
			<td colspan="3">150毫米氯化钠</td>
		</tr>
		<tr>
			<td rowspan="5">高盐缓冲器</td>
			<td colspan="3">0.1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1% 海卫 X-100</td>
		</tr>
		<tr>
			<td colspan="3">500毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20毫米三盐酸 pH8</td>
		</tr>
		<tr>
			<td colspan="3">500毫米氯化钠</td>
		</tr>
		<tr>
			<td rowspan="4">氯化缓冲区</td>
			<td colspan="3">250毫米氯化</td>
		</tr>
		<tr>
			<td colspan="3">1% NP-40</td>
		</tr>
		<tr>
			<td colspan="3">1毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">10毫米三盐酸盐 pH 值8</td>
		</tr>
		<tr>
			<td rowspan="2">洗脱缓冲器</td>
			<td colspan="3">1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">100 mM NaHCO3</td>
		</tr>
		<tr>
			<td>三 EDTA (TE) 缓冲器</td>
			<td colspan="3">50毫米三盐酸盐 pH 值8</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">1毫米 EDTA</td>
		</tr>
	</tbody>
</table>
表 1: 缓冲组合。

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Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

改进的小鼠骨骼肌染色质沉淀的协议

本文介绍了一种新的制备成年小鼠骨骼肌染色质的方法, 该方法适合于用染色质沉淀研究肌纤维的基因调控。

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

improved-protocol-for-chromatin-immunoprecipitation-from-mouse

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.4K 次观看.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. 本文介绍了一种新的制备成年小鼠骨骼肌染色质的方法, 该方法适合于用染色质沉淀研究肌纤维的基因调控。

视频: Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

从人体二尖瓣提取蛋白质的优化方案

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

科研

生物化学

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

从交联骨骼肌制备高质量核的基于过滤的方法进行染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

一种用于小数目造血干细胞的西方印迹协议

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

游泳运动员心肺评估的快速递增的绑带游泳最大协议

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

细菌中的RIBO-seq:NGS测序样本收集和库准备协议

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

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, ...