Name: measurement of protein import capacity of skeletal muscle mitochondria
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Description: Watch this Scientific Journal Video about Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria at JoVE.com

作者要感谢麦吉尔大学的G.C. Shore博士，华盛顿医学院的A. Strauss博士和拉筹伯大学的.M.T. Ryan博士最初捐赠了用于这项研究的表达质粒。这项工作得到了加拿大自然科学和工程研究委员会（NSERC）对D. A. Hood的资助。D. A. Hood也是加拿大细胞生理学研究主席的持有人。

这些实验中使用的所有动物都保存在约克大学的动物护理设施中。这些实验是根据加拿大动物护理委员会指南进行的，并得到了约克大学动物护理委员会的批准（许可证：2017-08）。
1. 从骨骼肌中分离出构肌下和肌原纤维间线粒体的功能分离
<ol><li>试剂制备：<ol>
<li>按照 表1所述准备所有缓冲液和培养基。</li>
		<li>将缓冲液设置为pH 7.4并储存在4°C（长达2周），?...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+regulates+PINK1+import+and+proteolytic+destabilization+by+PARL.">Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL.</a> The Journal of Cell Biology. 191 (5), 933-942 (2010).</li><li>Matsuda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1+stabilized+by+mitochondrial+depolarization+recruits+Parkin+to+damaged+mitochondria+and+activates+latent+Parkin+for+mitophagy.">PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy.</a> The Journal of Cell Biology. 189 (2), 211-221 (2010).</li><li>Fiorese, C. J., Schulz, A. M., Lin, Y. -. F., Rosin, N., Pellegrino, M. W., Haynes, C. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrion+plasticity+of+TOM+complex+assembly+in+skeletal+muscle+mitochondria+in+response+to+chronic+contractile+activity.">Mitochondrion plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity.</a> Mitochondrion. 12 (2), 305-312 (2012).</li><li>Singh, K., Hood, D. 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线粒体独特地依赖于核基因组和线粒体基因组的表达和协调，以在细胞内合成和扩增。然而，核基因组编码绝大多数（99%）的线粒体蛋白质组，这强调了蛋白质导入机制在支持线粒体生物发生方面的重要性。导入也是一个重要的信号传导事件，因为导入失败会促进未折叠蛋白质反应和/或线粒体自噬的启动15，16，26。由于导入?...

线粒体是存在于不同细胞类型复杂形态中的细胞器，并且被认为具有对细胞健康至关重要的越来越多的功能。因此，它们不能再仅仅被削减为产生能量的细胞器。线粒体是关键的代谢调节因子，细胞命运的决定因素和信号传导中枢，其功能可以作为整体细胞健康的有用指标。在骨骼肌细胞中，电子显微镜研究揭示了地理上不同的构体下（SS）和肌原纤维间（IMF）线粒体的存在，它们表现出一定程度的连接性1，2，3，4现在被认为是高度动态的，并且能够适应骨骼肌活动水平的变化，以及年龄和疾病。可以通过多种方式评估肌肉中的线粒体含量和功能5，6，并且已经应用了传统的细胞器分离方法来更好地了解线粒体的呼吸和酶促能力（Vmax），这与细胞环境的影响不同7，8。特别是，这些传统方法揭示了从构肌下和肌原纤维间区域分离的线粒体之间的微妙生化差异，掩盖了这些亚细胞区域代谢的可能功能意义8，9，10，11。
线粒体的生物发生是独特的，需要来自核和线粒体DNA的基因产物的贡献。然而，其中绝大多数来自细胞核，因为mtDNA转录仅导致13种蛋白质的合成。由于线粒体通常由参与不同代谢途径的>1000蛋白组成，因此细胞器的生物发生需要严格调节的方式将前体蛋白从细胞质基质导入和组装到各种线粒体亚室中，以维持适当的化学计量学和功能12，13。用于线粒体的核编码蛋白通常携带线粒体靶向序列（MTS），该序列将它们靶向细胞器并促进其亚区室定位。大多数基质结合蛋白含有可切割的N末端MTS，而那些发往线粒体膜外或内侧的蛋白通常具有内部靶向结构域14。导入过程由一组不同的通道进行，这些通道为进入细胞器提供了多种途径13。外膜（TOM）复合物的转座将前体从细胞质基质运送到膜间空间，在那里它们被内膜（TIM）复合物的转座体识别。该复合物负责将核编码的前体导入基质中，其中蛋白酶切割N端靶向预序列。发往外膜的蛋白质可以通过TOM复合物直接插入该膜中，而那些发往内膜的蛋白质则由TIM蛋白插入，特别是TIM22。在导入之后，蛋白质由常驻蛋白酶和伴侣进一步处理，并且通常结合形成更大的复合物，例如在电子传递链中发现的复合物。
线粒体蛋白导入本身也可以作为线粒体健康的测量，因为该过程依赖于膜电位的存在和ATP15形式的能量来源。例如，当膜电位消散时，蛋白激酶PINK1不能被细胞器吸收，这导致磷酸化信号，通过称为线粒体自噬的途径触发细胞器降解的开始16，17。在类似情况下，当导入受阻时，蛋白质ATF5不能进入细胞器，随后易位到细胞核，在那里它作为UPR基因表达上调的转录因子18，19。因此，测量蛋白质导入效率可以提供对细胞器健康状况的全面洞察，而基因表达响应可用于指示向细胞核的逆行信号传导的程度。
尽管它对线粒体的生物发生和一般的细胞健康具有明显的重要性，但哺乳动物线粒体的导入途径研究不足。在本报告中，我们描述了测量前体蛋白导入骨骼肌线粒体所涉及的具体步骤，并提供数据来说明导入系统对肌肉变化和废弃的适应性反应，说明蛋白质导入对骨骼肌适应性可塑性的贡献。

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			<td>0.2% BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>35S-methionine</td>
			<td>Perkin Elmer</td>
			<td>NEG709A500UC</td>
			<td>Purchase requires a valid radioisotope permit</td>
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			<td>ATP</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td></td>
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		<tr>
			<td>Blotting paper; Whatman 3MM CHR Paper</td>
			<td>Thermo Fisher</td>
			<td>05-714-5</td>
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			<td>Cassette for film</td>
			<td>Kodak</td>
			<td>Kodak Xomatic</td>
			<td></td>
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			<td>Centrifugation Tube</td>
			<td>Thermo Fisher</td>
			<td>3138-0050</td>
			<td></td>
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			<td>Chloroform</td>
			<td>Thermo Fisher</td>
			<td>C298-4</td>
			<td></td>
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		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>D9779-5G</td>
			<td></td>
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		<tr>
			<td>EDTA</td>
			<td>BioShop</td>
			<td>EDT002</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Dryer</td>
			<td>BioRad</td>
			<td>Model 583</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Drying Kit</td>
			<td>Sigma or BioRad</td>
			<td>Z377570-1PAK or OW-GDF-10</td>
			<td>Various options are commercially available through many companies, these are just as few examples.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>5350-1-40</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Avanti J-25 Centrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>IKA</td>
			<td>T25 Digital Ultra Turrex</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoamylalcohol, or 3-methylbutanol</td>
			<td>Sigma</td>
			<td>I9392</td>
			<td></td>
		</tr>
		<tr>
			<td>KAc</td>
			<td>BioShop</td>
			<td>POA301.500</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>M7G</td>
			<td>New England Biolab</td>
			<td>S1404S</td>
			<td>Dilute with 1000ul 20mM HEPES to make 1mM stock</td>
		</tr>
		<tr>
			<td>MgCl</td>
			<td>BioShop</td>
			<td>MAG510</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Thermo Fisher</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>BioShop</td>
			<td>MOP001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BioShop</td>
			<td>SOD001</td>
			<td></td>
		</tr>
		<tr>
			<td>NTP</td>
			<td>Thermo Fisher</td>
			<td>R0191</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Plasmid</td>
			<td>-</td>
			<td>-</td>
			<td>Donated from Dr. G. C. Shore, McGill University, Montreal, Canada; alternative available through Addgene, plasmid #71877</td>
		</tr>
		<tr>
			<td>pGEM4Z/hTom40 Plasmid</td>
			<td>-</td>
			<td>-</td>
			<td>Donated from Dr. M. T. Ryan, La Trobe University, Melbourne, Australia</td>
		</tr>
		<tr>
			<td>pGMDH Plasmid</td>
			<td>-</td>
			<td>-</td>
			<td>Donated from Dr. A. Strauss, Washington University School of Medicine</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma</td>
			<td>P4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyalcohol</td>
			<td>Sigma</td>
			<td>P3803</td>
			<td>Can also be made with the ratio provided</td>
		</tr>
		<tr>
			<td>Phosphorus Film</td>
			<td>Fujifilm</td>
			<td>BAS-IP MS 2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit reticulocyte lysate</td>
			<td>Promega</td>
			<td>L4960</td>
			<td>Avoid freeze-thaw; aliquot lysate upon arrival; amino acids are provided in the kit as well</td>
		</tr>
		<tr>
			<td>RNAsin</td>
			<td>Promega</td>
			<td>N2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor for High Speed Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>JA-25.50</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>BioShop</td>
			<td>SDS001.500</td>
			<td>Caution: harmful if ingested or inhaled, wear a mask.</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Bioshop</td>
			<td>SAA 304</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate</td>
			<td>VWR</td>
			<td>BDH9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium salicylate</td>
			<td>Millipore Sigma</td>
			<td>106601</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma</td>
			<td>S6021</td>
			<td></td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase</td>
			<td>Promega</td>
			<td>P1085</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td>Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S-2626</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioShop</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNA Polymerase</td>
			<td>Promega</td>
			<td>P2075</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop Centrifuge</td>
			<td>Thermo Fisher</td>
			<td>AccuSpin Micro 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Thermo Fisher</td>
			<td>A322-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BioShop</td>
			<td>TRS001</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of protein import capacity of skeletal muscle mitochondria

线粒体是关键的代谢和调节细胞器，决定能量供应以及细胞的整体健康状况。在骨骼肌中，线粒体存在于一系列复杂的形态中，从小的椭圆形细胞器到广泛的网状网络。 了解线粒体网如何随着各种刺激（如能量需求的变化）而膨胀和发展，长期以来一直是研究的主题。 这种生长或生物发生的一个关键方面是前体蛋白的导入，这些前体蛋白最初由核基因组编码，在细胞质基质中合成，并转移到各种线粒体亚室中。线粒体已经为这种导入过程开发了一种复杂的机制，涉及许多选择性的内膜和外膜通道，称为蛋白质导入机制（PIM）。导入线粒体取决于活膜电位和通过氧化磷酸化获得的细胞器衍生的ATP的可用性。因此，它的测量可以作为细胞器健康的衡量标准。PIM还在骨骼肌中表现出高水平的适应性可塑性，与细胞的能量状态紧密耦合。 例如，运动训练已被证明可以增加导入能力，而肌肉使用可以减少导入能力，这与线粒体含量标志物的变化相吻合。虽然蛋白质导入是线粒体生物发生和扩张的关键步骤，但该过程在骨骼肌中并未得到广泛研究。 因此，本文概述了如何使用骨骼肌中分离和功能齐全的线粒体来测量蛋白质导入能力，以促进对所涉及的方法的更多理解，并了解细胞器更新途径在运动，健康和疾病中的重要性。

我们已经广泛地说明了该协议是一种有效的测定方法，用于确定导入功能性和完整分离的骨骼肌线粒体的速率。与未经处理的条件相比，将典型的前体蛋白（如苹果酸脱氢酶（MDH））导入基质对膜电位敏感，因为它可以被一种呼吸链解聚剂valinomycin抑制（图2A）。当线粒体内膜和外膜在洗涤剂Triton X-100存在下溶解时，进口也会受到阻碍。导入过程对外部ATP的存在?...

Watch this Scientific Journal Video about Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria at JoVE.com

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

线粒体是关键的代谢细胞器，在骨骼肌中表现出高水平的表型可塑性。从细胞质基质中导入蛋白质是细胞器生物发生的关键途径，对于网状物的扩增和线粒体功能的维持至关重要。 因此，蛋白质进口是细胞健康的晴雨表。

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, ...

线粒体严重依赖核基因组，尽管它们有自己的DNA。因此，需要一个高度管制的进口机制。这种方法有助于研究该过程以及它如何适应外部刺激。这是唯一可用于定量评估蛋白质导入线粒体各个亚区室的生化技术。线粒体的生存能力与各种疾病状态有关，因此了解蛋白质进口如何被调节可能有助于靶向线粒体的新治疗方法。在隔离期间需要格外小心，以确保线粒体的活力和完整性。彻底切碎组织并轻轻地重复所有后续颗粒是必要的。演示该程序的将是Ashley Oliveira，我实验室的博士生。要开始将线粒体从骨骼肌中分离出来，请从肌肉中除去脂肪和结缔组织，并在预冷的手表玻璃上切碎组织，直到它成为均匀的浆液。将切碎的组织放入预冷的50毫升塑料离心管中，并记录确切的重量。然后，用含有ATP的缓冲液1将切碎的组织稀释十倍。使用八毫米双刀片均质机以9.8赫兹的功率输出对肌肉样品进行均质化10秒，确保没有可见的肌肉块残留。在4摄氏度下以9， 000x G旋转样品10分钟后，将沉淀小心地重悬于约95微升的重悬培养基中。在丢弃上清液之前离心样品，并使用P-200移液管用大约180微升的重悬培养基轻轻重悬沉淀。对于体外翻译，准备足够的翻译反应混合物，为每个线粒体样品提供20微升，用于导入实验和翻译通道。将反应物置于30摄氏度下孵育30分钟。翻译反应开始后15分钟，将90微克线粒体等分到无菌的1.5毫升管中，并在30摄氏度下预孵育10分钟。在一组新的无菌1.5毫升管中，将75微克线粒体与18微升的平移反应混合，并将管子在30摄氏度下孵育所需的时间。将翻译反应的剩余体积保持在冰上。为了在适当的孵育时间后终止进口反应，从30摄氏度取出试管将其放在冰上，然后小心地将进口反应转移到带有蔗糖垫的管顶上。用蔗糖垫在17， 000x G下在4摄氏度下离心样品15分钟。使用P-1000移液器，小心地除去上清液而不干扰沉淀。要导入外膜，使用Tom40进行反应，并将沉淀重悬于50微升新鲜制备的0.1摩尔碳酸钠中，并在冰上孵育30分钟。孵育后，将样品在4摄氏度下以14， 000x G离心五分钟。接下来，按照手稿中所述为SDS-PAGE电泳准备样品。通过将剩余翻译反应的3微升与37微升裂解缓冲液和5微升样品染料混合，准备对照平移通道。将样品在95摄氏度下煮沸五分钟，然后以低速轻轻旋转，以避免线粒体成粒。将样品涂敷到SDS聚丙烯酰胺凝胶中后，在通风橱中将凝胶在5%三氯乙酸或TCA中煮沸五分钟，并连续搅拌。然后，将凝胶置于双蒸馏水中，以每分钟50次旋转的速度旋转一分钟。将凝胶在旋转板上以每分钟50次旋转的速度在10毫摩尔Tris中洗涤5分钟。为了沉淀蛋白质，用足够体积的单摩尔水杨酸洗涤凝胶，以将凝胶覆盖在旋转板上30分钟。要使凝胶脱水，请将一大张吸墨纸放在凝胶干燥机的多孔床上，并在将要应用凝胶的区域放置一张纸巾。然后，将11厘米×9厘米的吸墨纸切成11厘米，并将纸放在纸巾上。使用第二张11厘米×9厘米的吸墨纸从容器中舀出凝胶，然后将凝胶平放在第一块的顶部。在凝胶上放一块稍大的塑料，确保包装下没有折痕或气泡。然后，将凝胶干燥器的塑料盖放在包装的顶部。打开真空吸尘器。通过抬起塑料盖的一角并等待盖子重新密封，确保塑料盖已形成密封。关闭凝胶干燥器运行90分钟，从30摄氏度开始逐渐达到80摄氏度，并在运行结束时返回30摄氏度。将凝胶包裹在干燥过程中使用的保鲜膜中。一旦脱水，凝胶会凝固并感觉像薄纸一样。将脱水凝胶置于顶部有磷膜的盒中。在使用任何能够进行磷成像的合适成像仪使用放射自显像仪将凝胶可视化之前，先将胶片暴露24小时。代表性分析显示了苹果酸脱氢酶（MDH）在构肌下和肌原纤维间线粒体中的正常导入速率，以及前体MDH的翻译产物。加伐霉素抑制了MDH蛋白导入SS和IMF线粒体的基质。类似地，洗涤剂Triton X由于内膜的增溶而抑制了MDH导入到两个亚组分中。对蛋白质进口的估计是在增加持续时间的情况下进行的，随后的数据表明，进口是一个时间依赖的过程，SS和IMF线粒体具有不同的进口率或能力。当大鼠受到电刺激时，与对照组相比，长期刺激动物导入外膜的Tom 40肌肉含量更高。鸟氨酸转氨甲酰酶或OCT导入到线粒体基质中，在孵育的每个时间点都增加，导致长期刺激的肌肉的线粒体增加1.4倍。蛋白质导入与线粒体含量指数呈正相关，并通过COX活性进行评估。在研究线粒体介导的细胞凋亡时，Bax和Bak双敲除动物表现出减少蛋白质导入线粒体基质。经过六周的自愿轮跑，挽救了双敲除动物的进口缺陷。重要的是要考虑变量，例如进口反应的持续时间以及要进口的蛋白质。细胞质级分可以掺入反应中，以了解胞质因子如何影响输入速率。或者，可以在孵育前对蛋白质进行修饰，以了解蛋白质如何选择性地导入。这种技术可以帮助理解可能以各种疾病状态出现的线粒体肌病的复杂病因。

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

Unit 1

Intracellular Compartments and Protein Sorting

SCIENTISTS IN ACTION

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

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 ...

科研

生物化学

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

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

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

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

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

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 ...

Studying Protein Import into Chloroplasts Using Protoplasts

原生质体在叶绿体中导入蛋白质的研究

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

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 ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...