这项工作部分得到了堪萨斯州INBRE、P20GM103418、李和齐诺维耶娃以及R35GM124828和齐诺维耶娃的支持。我们感谢韩敏慷慨地分享抗ANI-1抗体。这项工作过程中使用的一些菌株由国家卫生研究院研究基础设施项目办公室（P40 OD010440）资助的 卡诺哈布迪蒂斯 遗传学中心（CGC）提供。

1. 蠕虫样本收集
<ol><li>种子混合阶段或同步13蠕虫在 NGM 固体板在所需的温度，并允许蠕虫生长，直到所需的阶段。有关基本的C.elegans生长和维护，请参阅斯蒂埃纳格尔等人和波尔塔德拉里瓦等人14，13。</li>
	<li>通过用 M9 缓冲器清洗蠕虫板，在 15 mL 锥形离心管中收集蠕虫。</li>
	<li>在室温 （RT） 下在 400 x g 下离心 2 分钟，将蠕虫丸化，然后丢弃超自然物。 注：用于提取物制备的蠕虫颗粒大小在 100 μL 和 500 μL 之间。建议进行下游免疫沉淀实验，并推荐300微升的包装蠕虫颗粒，通常产生约4.5毫克的总蛋白质，而500微升颗粒将产生约7.5毫克的总蛋白质。</li>
	<li>使用 M9 缓冲区执行额外的 3–5 洗涤（参见 表 1），或直到超自然物不再多云。</li>
	<li>使用ddH2O进行最后一次洗涤。</li>
	<li>将松散的蠕虫颗粒移动到 1.5 mL 微中枢管，并在 RT 以 400 x g 的速度旋转 2 分钟。丢弃剩余的超自然物，获取包装好的蠕虫颗粒，然后进行提取准备。 注：协议可在此处暂停。蠕虫颗粒可立即冻结在液氮中，储存在 -80 °C 或液氮中。请注意，蠕虫颗粒只能解冻一次，不能重新冻结。</li>
</ol>2. 提取蠕虫颗粒的制备
注意：提取物准备应在冰上或在4°C下进行。
<ol><li>如果冻结，在冰上解冻蠕虫颗粒。 注意：如果在样品采集过程中未获得所需的 300 μL 包装蠕虫颗粒大小，则可以组合多个较小的颗粒，直到存在足够的材料以进一步提取。</li>
	<li>添加等量的冰冷2倍裂解缓冲器（60米重， pH = 7.4， 100 mM 氯化钾， 0.1% 三通 X， 4 mM 氯化镁， 10% 甘油， 2 mM DTT 与 RNase 抑制剂， 蛋白酶抑制剂， 和磷酸盐抑制剂; 见 表 1）和涡流或移液器上下混合.将管子向下旋转，以收集管底部的混合物。</li>
	<li>将混合物移入含有金属珠子的 1.5 mL RNase 无管中（参见 材料表），并将样品放入珠磨同质化剂（见 材料表）中，共度 4 °C。 确保管盖拧紧，样品在均质器内保持平衡。</li>
	<li>以最高速度（设置 12）将样品同质化 4 分钟。</li>
	<li>从珠子中取出样品，放入新的 1.5 mL 微中微管中。或者，使用同质化剂提供的磁铁可用于从样品中去除珠子。</li>
	<li>在 4 °C 下以 19，000 x g 的速度将提取物旋转 20 分钟，以澄清蛋白质提取物。</li>
	<li>将超自然物转移到冰上一个新鲜的 1.5 mL 管中，同时避免在样品顶部形成的白色多云沉淀物的随身行李。超自然人现在是澄清的提取物。 注：保存澄清提取物的10μL以确定蛋白质总浓度。</li>
	<li>立即使用提取物进行以下实验或将提取物闪冻在液氮中，储存在 -80 °C。 注：协议可能会在这里暂停。提取物可在超低温下储存（-80°C冰柜或液氮约6个月）。冷冻提取物可以解冻一次，不能重新冷冻。</li>
	<li>使用与洗涤剂兼容的蛋白质浓度检测套件（见材料表）确定提取物的总蛋白质浓度（参见 材料表）。</li>
</ol>3. 免疫沉淀
注：提取准备的所有免疫沉淀步骤应在冰上或4°C进行。 建议每次免疫沉淀使用2毫克总蛋白质。然而，已经成功地进行了总蛋白质0.8-1毫克的免疫沉淀。始终使用新鲜或新鲜解冻的蛋白质提取物。下列协议概述从总蛋白质的2毫克或单一免疫沉淀实验进行免疫沉淀。对于多个样本，或者如果使用了不同量的蛋白质提取物，珠子和抗体的数量可能会相应增加或减少。
<ol><li>将蛋白质提取物放在冰上或解冻。样品可用冰冷的1倍裂解缓冲器稀释为10毫克/mL或5毫克/mL（见表1）。</li>
	<li>通过倒置来恢复磁珠，并将 50% 珠子悬浮的 150 μL 传输到 1.5 mL 管中。将冰上的珠子磁化1分钟或直到溶液清晰为止。丢弃超自然人。</li>
	<li>从磁性支架中取出管子，使用 2 卷（即 300 μL）珠子浆洗 1 倍裂解缓冲器中的珠子。重复洗涤2次，共洗三次。</li>
	<li>将珠子重新用150微L的冰冷溶解缓冲器。</li>
	<li>将珠浆中的 75 μL 转移到 2 毫克蛋白质提取物中，并在 4 °C 下孵育 1 小时，轻轻搅拌。将剩余的珠子悬架保存在冰上供以后使用。 注意：此步骤是为了减少免疫沉淀过程中与珠子结合的非特异性蛋白质。</li>
	<li>将带样品的管子放在冰上的磁性支架上1分钟，或直到珠子完全磁化，样品清晰。将超自然人转移到新的 1.5 mL 管中;不要打扰珠子。这是预先清除的蛋白质化解。将 10% 的样本保存在西方污点分析中。</li>
	<li>在预先清除的番茄酱中加入 20 μg 的亲和纯化抗体，在 4 °C 下孵育 1 小时，轻轻搅拌。 注：用于免疫沉淀的抗体量是特定于抗体和蛋白质的，应经经验确定，以确保目标蛋白的有效免疫沉淀。</li>
	<li>将预洗珠悬架（步骤 3.5） 的剩余 75 μL 添加到抗体/lysate 混合物中，在 4 °C 下以温和搅拌孵育 1 小时。</li>
	<li>将管子放在冰上的磁性支架上1分钟，或直到珠子完全磁化，样品清晰。保存超自然的西方污点分析（可选）。</li>
	<li>在 450 μL 的洗涤缓冲区（30 mM HEPES，pH = 7.4，100 mM 氯化钾、0.1% Triton X、2 mM 氯化镁、10% 甘油、1 mM DTT;见 表 1）中清洗含有免疫沉淀物 3 倍的珠子。 注意：如果选择更严格的洗涤条件，可以进行额外的洗涤。</li>
	<li>在加载到 SDS-PAGE 凝胶之前，在 95 °C 煮沸 5 分钟之前，将珠子颗粒重新加入 20 μL 的 2x SDS/BME 蛋白凝胶加载缓冲器（参见 材料表），然后去自然。或者，变性样品可以在-20°C下储存数月。 注意：如果需要，可以保存一部分珠免疫沉淀剂，用于下游RNA隔离。</li>
</ol>4. IP 样品的西方污点检测
<ol><li>将 IP 样本加载到 SDS-PAGE 凝胶上（参见 材料表）。避免转移珠子，在样品吸气前将 IP 管放在磁性支架上 1 分钟。</li>
	<li>执行西印15，16和抗体染色16与以下修改：稀释LG-1抗体17 1：500在5%非脂干牛奶（NFDM）：稀释HRPK-1抗体2 1：1，000在5%NFDM中;并在5%NFDM中稀释ANI-1抗体18 1：10，000。根据制造商的说明使用了二次抗体（见材料表）。</li>
	<li>检测带有基于 HRP 的化学发光带（参见 材料表）。</li>
</ol>

<ol>
	<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+AP-MS-+and+BioID-compatible+MAC-tag+enables+comprehensive+mapping+of+protein+interactions+and+subcellular+localizations.">An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations.</a> Nature Communications. 9 (1), 1188-1216 (2018).</li><li>Li, L., Veksler-Lublinsky, I., Zinovyeva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HRPK-1,+a+conserved+KH-domain+protein,+modulates+microRNA+activity+during+Caenorhabditis+elegans+development.">HRPK-1, a conserved KH-domain protein, modulates microRNA activity during Caenorhabditis elegans development.</a> PLoS Genetics. 15 (10), 1008067 (2019).</li><li>Zinovyeva, A. Y., Veksler-Lublinsky, I., Vashisht, A. A., Wohlschlegel, J. A., Ambros, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+ALG-1+antimorphic+mutations+uncover+functions+for+Argonaute+in+microRNA+guide+strand+selection+and+passenger+strand+disposal.">Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (38), 5271-5280 (2015).</li><li>Hammell, C. M., Lubin, I., Boag, P. R., Blackwell, T. K., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nhl-2+Modulates+microRNA+activity+in+Caenorhabditis+elegans.">nhl-2 Modulates microRNA activity in Caenorhabditis elegans.</a> Cell. 136 (5), 926-938 (2009).</li><li>Zou, Y., 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=Developmental+decline+in+neuronal+regeneration+by+the+progressive+change+of+two+intrinsic+timers.">Developmental decline in neuronal regeneration by the progressive change of two intrinsic timers.</a> Science. 340 (6130), 372-376 (2013).</li><li>Zanin, E., Dumont, J., 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=Affinity+purification+of+protein+complexes+in+C.+elegans.">Affinity purification of protein complexes in C. elegans.</a> Methods in Cell Biology. 106, 289-322 (2011).</li><li>Bhaskaran, S., 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=Breaking+Caenorhabditis+elegans+the+easy+way+using+the+Balch+homogenizer:+an+old+tool+for+a+new+application.">Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: an old tool for a new application.</a> Analytical Biochemistry. 413 (2), 123-132 (2011).</li><li>Kohl, 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=Plate-based+Large-scale+Cultivation+of+Caenorhabditis+elegans:+Sample+Preparation+for+the+Study+of+Metabolic+Alterations+in+Diabetes.">Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes.</a> Journal of Visualized Experiments. (138), e58117 (2018).</li><li>Larance, M., Bailly, A. P., 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=Stable-isotope+labeling+with+amino+acids+in+nematodes.">Stable-isotope labeling with amino acids in nematodes.</a> Nature Methods. 8 (10), 849-851 (2011).</li><li>Ding, L., Han, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GW182+family+proteins+are+crucial+for+microRNA-mediated+gene+silencing.">GW182 family proteins are crucial for microRNA-mediated gene silencing.</a> Trends in Cell Biology. 17 (8), 411-416 (2007).</li><li>Ding, L., Spencer, A., Morita, K., Han, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Developmental+Timing+Regulator+AIN-1+Interacts+with+miRISCs+and+May+Target+the+Argonaute+Protein+ALG-1+to+Cytoplasmic+P+Bodies+in+C.+elegans.">The Developmental Timing Regulator AIN-1 Interacts with miRISCs and May Target the Argonaute Protein ALG-1 to Cytoplasmic P Bodies in C. elegans.</a> Molecular Cell. 19 (4), 437-447 (2005).</li><li>Jannot, G., Vasquez-Rifo, A., Simard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Argonaute+Pull-Down+and+RISC+Analysis+Using+2&#8217;-O-Methylated+Oligonucleotides+Affinity+Matrices.">Argonaute Pull-Down and RISC Analysis Using 2&#8217;-O-Methylated Oligonucleotides Affinity Matrices.</a> Methods in Molecular Biology. 725, 233-249 (2011).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Ceron, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> Journal of Visualized Experiments. (64), e4019 (2012).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook: the Online Review of C. elegans Biology. , 1-11 (2006).</li><li>Gallagher, S., Chakavarti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblot+analysis.">Immunoblot analysis.</a> Journal of Visualized Experiments. (16), e759 (2008).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> Journal of Visualized Experiments. (44), e2359 (2010).</li><li>Zinovyeva, A. Y., Bouasker, S., Simard, M. J., Hammell, C. M., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+conserved+residues+of+the+C.+elegans+microRNA+Argonaute+ALG-1+identify+separable+functions+in+ALG-1+miRISC+loading+and+target+repression.">Mutations in conserved residues of the C. elegans microRNA Argonaute ALG-1 identify separable functions in ALG-1 miRISC loading and target repression.</a> PLoS Genetics. 10 (4), 1004286 (2014).</li><li>Zhang, L., 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=Systematic+Identification+of+C.+miRISC+Proteins,+miRNAs,+and+mRNA+Targets+by+Their+Interactions+with+GW182+Proteins+AIN-1+and+AIN-2.">Systematic Identification of C. miRISC Proteins, miRNAs, and mRNA Targets by Their Interactions with GW182 Proteins AIN-1 and AIN-2.</a> Molecular Cell. 28 (4), 598-613 (2007).</li><li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+Window+into+Biology:+A+Primer+on+Caenorhabditis+elegans.">A Transparent Window into Biology: A Primer on Caenorhabditis elegans.</a> Genetics. 200 (2), 387-407 (2015).</li><li>Farboud, B., 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=Enhanced+Genome+Editing+with+Cas9+Ribonucleoprotein+in+Diverse+Cells+and+Organisms.">Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.</a> Journal of Visualized Experiments. (135), e57350 (2018).</li><li>Dickinson, D. J., Goldstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Based+Methods+for+Caenorhabditis+elegans+Genome+Engineering.">CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering.</a> Genetics. 202 (3), 885-901 (2016).</li></ol>

作者没有什么可透露的。

C. elegans是研究细胞、分子和发育生物学基础问题的优秀模型。除了作为基因模型系统的力量外，C.elegans还适应生化方法，包括但不限于蛋白质免疫沉淀和共免疫沉淀。在进行免疫预测实验时，一个潜在的障碍是缺乏与感兴趣的蛋白质特有的抗体。如果没有抗体，可以生成自定义多克隆或单克隆抗体。然而，最近基因组编辑技术的创新使研究人员能够迅速引入突变或标记内源性C.elegans基因20，21，促进研究，解开基因和编码蛋白质之间的遗传，功能和物理相互作用。具体来说，CRISPR/Cas9介导的C.elegans基因标记在内源性位点减少了免疫预测实验对抗体可用性的依赖性，使共免疫介质实验更加可行。C. elegans基因可以标记各种标签，从荧光标签（如 GFP 或 mCherry）到小型标签（如 FLAG 和 HA）。识别这些标签的抗体在商业上是现成的，通过免疫沉淀方法促进蛋白质-蛋白质相互作用的研究。
图1中概述的所述协议可用于少量样品或放大，允许一次最多 24 个样品制备。虽然通过免疫沉淀进行的蛋白质-蛋白质相互作用的初始特征通常在正常生长条件下的野生类型背景中完成，但后续研究往往需要测试各种遗传背景或不同生长条件下的蛋白质-蛋白质相互作用。同时准备多个提取物的能力节省了时间，更重要的是，确保了不同样品之间的提取准备一致性。始终需要负控制，理想的控制是编码为免疫沉淀蛋白的基因中的零突变（如图3和图 4）。
这种提取协议允许从 C.elegans 样品快速蛋白质提取制备，并可与硅珠基同质化8相媲美。珠子均质化一般可以扩展到多个同时样品制剂使用各种珠磨同质化剂或类似的设备。然而，一些更经济的珠子磨合金可能减少可以同时处理的样品数量。或者，所提出的提取协议与基于 dounce 的提取物制备兼容，后者代表一种经济的替代方案。虽然不同的珠磨同质化剂没有经过测试，但只要 C.elegans 样品完全中断，大多数都可能与这种蛋白质提取协议兼容。
如前所介绍，此提取物制备协议与多个下游实验兼容，包括蛋白质免疫沉淀2和 microRNA 下拉12，并允许下游收集蛋白质和RNA组件。它还有效地提取核蛋白和细胞质蛋白（图2、图3和图4）。同样，提出的免疫沉淀协议允许RNA与蛋白质相关的免疫沉淀物隔离。虽然免疫沉淀协议最初是为了识别LG-1蛋白质相互作用者而开发的，但该方法可以适应测试任何感兴趣的蛋白质之间的相互作用。事实上，用于免疫沉淀的免疫沉淀条件同样有效，用于免疫预测的LG-1（图3）和HRPK-1（图4）。该协议是RNA结合蛋白免疫净化的良好起点。然而，应该指出，对于其他感兴趣的蛋白质，缓冲成分可能需要一些变化。这些变化可能取决于感兴趣的蛋白质的物理和生化特性，并且必须逐案实施。
一旦目标蛋白（这里，ALG-1或HRPK-1）被免疫，西式印迹可用于测试特定蛋白质相互作用者的共免疫沉淀。
或者，对共纯免疫沉淀物进行质谱分析，以确定所有假定相互作用的蛋白质。然后，可以在各种遗传背景或条件下检查已确认的共免疫沉淀相互作用，以确定特定相互作用的潜在调节。例如，为了确定 hrpk-1 是否在LG-1/AIN-1 miRISC组装中发挥作用，ALG-1-AIN-1共谋在野生类型背景和HRPK-1（图3）的情况下进行了评估。 hrpk-1 被发现可为 ALG-1/AIN-1 交互2 （图 3） 提供可有可无。此外，CRISPR/Cas9基因组编辑技术可用于在感兴趣的蛋白质中产生单点或域删除突变。重新测试生成突变体与其蛋白质相互作用器共生的能力，可以揭示哪些领域或残留物调解物理相互作用。这种未来的研究可以产生关于蛋白质功能和调节机制的宝贵信息。这些方法结合 了C.elegans 遗传学的力量，可以为指导动物发育和细胞功能的基本分子过程提供重要的见解。

识别感兴趣的蛋白质的巨分子相互作用可能是了解更多关于其功能的关键。免疫沉淀和共免疫沉淀实验可用于通过大规模蛋白质组学方法1识别蛋白质的整个相互作用，或具体测试蛋白质与假想相互作用者共生的能力。在C.elegans中，这两种方法都被成功地用于更多地了解各种蛋白质的活性，包括那些与微RNA紧密结合来调节基因表达2、3、4的蛋白质。共免疫沉淀实验具有测试其原生细胞环境中蛋白质-蛋白质相互作用的优势，但提取制备可能具有挑战性且耗时。需要对样品进行有效的裂解，但必须小心尽量减少蛋白质相互作用的破坏。方法，如调5，声波6，巴尔奇同质化7，和氧化硅珠-同质化8，9已经被用来成功地准备C.elegans总蛋白质提取物。这些方法，除了氧化锌珠同质化，在可以同时处理的样品数量方面有限制。提出是一种替代方法，可以很容易地扩大，使高通量，快速蛋白质提取制剂制备从C.elegans样品，然后共同免疫沉淀。具体来说，该方法一次最多可准备24个样品，大大缩短了提取准备所需的时间。相比之下，通常一次只允许一次准备一个样品。这种提取方法可用于准备从C.elegans的任何发育阶段提取物。
描述的是动物样本采集、提取准备、免疫沉淀和西式印迹数据的呈现的分步过程，以确认蛋白质提取和检测成功的兴趣共同免疫预测蛋白。为了证明该议定书的有效性，在1）微RNA阿戈纳特ARG-1和AIM-1（GW182同源）之间进行了两次共免疫预测实验：和 2） ALG-1 和 HRPK-1，新识别的 ALG-1 交互器2。ALG-1和AISE-1是核心蛋白质，组成微RNA诱导的沉默复合物（miRISC），这两种蛋白质之间的相互作用是众所周知的10，11。提取物制备方案在LG-1-AIN-1联合免疫沉淀实验中有效。该协议还成功地确认了LG-1与其新确定的交互体HRPK-12之间的相互作用。
总之，手稿描述了一个 C.elegans 提取准备协议，可以放大到同时处理24个样本以及一个共同免疫预测协议，可用于识别蛋白质之间的新或确认假想的相互作用。提取物制备协议与一些下游实验兼容，包括蛋白质免疫沉淀2和微RNA下拉12。此外，免疫沉淀协议可以适应测试任何两种或两种以上内源性、内源性标记或在各种遗传背景中过度表达的 C.elegans 蛋白之间的相互作用。

<table border="0" cellpadding="0" cellspacing="0" style="width:989px;" width="989">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">15 mL tube</td>
			<td style="width:195px;">VWR</td>
			<td style="width:119px;">89039-664</td>
			<td style="width:213px;">STEP 1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x Laemmli Sample Buffer</td>
			<td>BioRed</td>
			<td>1610737</td>
			<td>STEP 3.11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8211;20% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td>BioRed</td>
			<td>4561096</td>
			<td>STEP 4.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">anti-AIN-1 monoclonal antibody</td>
			<td style="width:195px;">custom generated</td>
			<td style="width:119px;">n/a</td>
			<td>STEP 4.2, see ref. Zhang et al. 2007</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">anti-ALG-1 monoclonal antibody</td>
			<td style="width:195px;">custom generated by PRF&#38;L</td>
			<td style="width:119px;">n/a</td>
			<td>STEP 4.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">anti-HRPK-1 monoclonal antibody</td>
			<td style="width:195px;">custom generated by PRF&#38;L</td>
			<td style="width:119px;">n/a</td>
			<td>STEP 4.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bullet Blender Storm Homogenizer</td>
			<td>MidSci</td>
			<td>BBY24M</td>
			<td>STEP 2.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">DL-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td style="width:119px;">D9779-5G</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads Protein A for Immunoprecipitation</td>
			<td>Thermo Fisher</td>
			<td>10002D</td>
			<td>STEP 3.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2 Magnet</td>
			<td>Thermo Fisher</td>
			<td>12321D</td>
			<td>STEP 3.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free protease inhibitors</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">GFP antibody (FL)</td>
			<td style="width:195px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-8334</td>
			<td>Figure 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Glycerol</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">G33-500</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Goat Anti-Rabbit Secondary Antibody, HRP</td>
			<td style="width:195px;">BioRed</td>
			<td style="width:119px;">1662408</td>
			<td>STEP 4.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Goat anti-Rat IgG (H+L) Secondary Antibody, HRP</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">31470</td>
			<td>STEP 4.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">HEPES</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">H4034-500G</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LICOR WesternSure PREMIUM Chemiluminescent Substrate, 100 mL Kit</td>
			<td>LI-COR</td>
			<td>926-95000</td>
			<td>STEP 4.3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:463px;">Magnesium chloride hexahydrate ACS</td>
			<td style="width:195px;">VWR</td>
			<td style="width:119px;">VWRV0288-500G</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Magnesium Sulfate Anhydrous</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">M65-500</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Microcentrifuge Tubes, 1.5 mL</td>
			<td style="width:195px;">VWR</td>
			<td style="width:119px;">20170-333</td>
			<td>STEP 1.6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 wild type</td>
			<td>CGC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Navy RINO RNA Lysis Kit 50 pack (1.5 mL)</td>
			<td>MidSci</td>
			<td>NAVYR1-RNA</td>
			<td>STEP 2.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphatase inhibitor cocktail 2</td>
			<td>Sigma</td>
			<td>P5726-1ML</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphatase inhibitor cocktail 3</td>
			<td>Sigma</td>
			<td>P0044-1ML</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Potassium Chloride</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">P217-500</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Potassium phosphate monobasic</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">P285-3</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RC DC Protein Assay Kit I</td>
			<td>BioRed</td>
			<td>5000121</td>
			<td>STEP 2.9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseOUT Recombinant Ribonuclease Inhibitor</td>
			<td>Thermo Fisher</td>
			<td>10777019</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Sodium Chloride</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">S271-500</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Sodium Phosphate Dibasic Anhydrous</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">S374-500</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">TritionX-100</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">X100-500ML</td>
			<td>Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UY38 hrpk-1(zen17)</td>
			<td>available upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">VT1367 col-19::gfp(maIS105)</td>
			<td>available upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VT3841 alg-1(tm492)</td>
			<td>available upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protein extract preparation and co-immunoprecipitation from caenorhabditis elegans

共免疫沉淀方法常用于研究蛋白质-蛋白质相互作用。确认假定蛋白-蛋白质相互作用或识别新的相互作用，可以提供有关感兴趣的蛋白质功能的宝贵信息。一些传统的提取制备方法往往需要劳动密集型和耗时的技术。在这里，使用珠磨同质化剂和金属珠子的改良提取物制备协议被描述为传统蛋白质制备方法的快速替代品。这种提取物制备方法与下游共免疫沉淀研究相容。例如，该方法用于成功共免疫C.elegans 微RNA阿戈纳特ARG-1和两个已知的ARG-1相互作用器：AIN-1和HRPK-1。该协议包括动物样本采集、提取物制备、提取物澄清和蛋白质免疫沉淀的描述。所述协议可以适应测试任何两种或两种以上内源性、内源性标记或在各种遗传背景中过度表达的 C. elegans 蛋白质之间的相互作用。

此协议（图1中计划）成功用于获取C.elegans总蛋白质提取物（图2），用于几个蛋白质2（图3和图4）的下游免疫沉淀。所呈现的珠磨同质化协议在总蛋白质提取方面可与基于豆类的方法（图2）相媲美，并有效提取核（COL-19：：GFP（NLS）（图2）和细胞质蛋白（图3和图4）。同时提取了多个不同大小的样本（图2）。阿戈纳特蛋白与GW182蛋白质家族的成员相互作用，形成与目标信使RNA结合并抑制其表达10的miRISC。图3显示，与先前的报告11、17一致，成功共免疫了核心MIRISC组件LG-1和AISE-1。最近，人们努力确定ArgonauteALG-1 3的其他蛋白质相互作用体，以便更多地了解微RNA生物生成和活性如何被辅助因素调节。RNA结合蛋白HRPK-1在LG-1免疫渗透3中被发现。这种相互作用最近在互惠HRPK-1免疫沉淀实验2中得到证实。所提交的提取物和免疫沉淀协议成功地在HRPK-1特异性共免疫预测（图4）中回收了LG-1。此外，ALG-1-AIN-1相互作用在各种遗传背景中进行了测试，HRPK-1被证明对LG-1/AIN-1miRISC组件2（图3）是不必要的。提供补充数字，以显示完整的膜探测（补充图1）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61243/61243fig1.jpg"> 图1：C.elegans提取制备和免疫沉淀的工作流程示意图。<a href="https://www.jove.com/files/ftp_upload/61243/61243fig1large.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/61243/61243fig2.jpg"> 图2：西方对核局部GFP（COL-19：：GFP）的污点比较，250μL和100μL蠕虫颗粒中由杜塞准备和同质化样品的水平。<a href="https://www.jove.com/files/ftp_upload/61243/61243fig2large.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/61243/61243fig3.jpg"> 图3：GW182同源AIM-1与LG-1共同免疫。 ALG-1和AIM-1蛋白的西方印迹在LG-1免疫渗透。ALG-1/AIN-1共同免疫接种没有受到 hrpk-1 缺乏影响。输入=IP的10%。 <a href="https://www.jove.com/files/ftp_upload/61243/61243fig3large.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/61243/61243fig4.jpg"> 图4：ALG-1与HRPK-1共免疫。 显示 HRPK-1 和 HRPK-1 免疫沉淀剂中的 HRPK-1 和 ALG-1 的西方印迹。输入=IP的10%。*表示抗体重链。 <a href="https://www.jove.com/files/ftp_upload/61243/61243fig4large.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"><tbody>
<tr>
<td colspan="2">M9 缓冲区 （1 L）</td>
		</tr>
<tr>
<td>KH2PO4
</td>
			<td>3 g</td>
		</tr>
<tr>
<td>娜2HPO4
</td>
			<td>6 g</td>
		</tr>
<tr>
<td>纳克</td>
			<td>5 克</td>
		</tr>
<tr>
<td>1 M 毫克SO4
</td>
			<td>1升</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>最多1升</td>
		</tr>
<tr>
<td colspan="2">2x 裂解缓冲区 （5 mL）</td>
		</tr>
<tr>
<td>赫佩斯 （pH 7.4）</td>
			<td>200μL</td>
		</tr>
<tr>
<td>2 M 千克</td>
			<td>250μL</td>
		</tr>
<tr>
<td>10% 特里顿克斯</td>
			<td>100μL</td>
		</tr>
<tr>
<td>1 M 毫克2
</td>
			<td>20 微升</td>
		</tr>
<tr>
<td>100% 甘油</td>
			<td>1升</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>高达5升</td>
		</tr>
<tr>
<td>添加新鲜：</td>
			<td></td>
		</tr>
<tr>
<td>1兆分</td>
			<td>20 微升</td>
		</tr>
<tr>
<td>无EDTA蛋白酶抑制剂</td>
			<td>1 片</td>
		</tr>
<tr>
<td>磷酸盐抑制剂鸡尾酒 2</td>
			<td>100μL</td>
		</tr>
<tr>
<td>磷酸盐抑制剂鸡尾酒 3</td>
			<td>100μL</td>
		</tr>
<tr>
<td colspan="2">1x 裂解缓冲区</td>
		</tr>
<tr>
<td colspan="2">稀释2倍裂解缓冲器，等量为ddH20。</td>
		</tr>
<tr>
<td colspan="2">1x 洗涤缓冲区 10 mL）</td>
		</tr>
<tr>
<td>赫佩斯 （pH 7.4）</td>
			<td>300μL</td>
		</tr>
<tr>
<td>2 M 千克</td>
			<td>500μL</td>
		</tr>
<tr>
<td>10% 特里顿克斯</td>
			<td>100μL</td>
		</tr>
<tr>
<td>1 M 毫克2
</td>
			<td>20 微升</td>
		</tr>
<tr>
<td>100% 甘油</td>
			<td>1升</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>高达10升</td>
		</tr>
<tr>
<td>1兆分</td>
			<td>20 微升（添加新鲜）</td>
		</tr>
</tbody></table>表1：食谱
补充图1。 显示用于生成图 2-4 的完整探测西印膜。（A） 图 2 的探寻膜。请注意，膜被切割，以便同时探测 GFP 和图布林，减少整体污点大小。（B） 图3的探寻膜。（C） 图3的探寻膜。*表示抗体重链。 <a href="https://www.jove.com/files/ftp_upload/61243/Supplemental_Figure_1.pdf" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans at JoVE.com

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

该方法描述了从 卡诺哈布迪蒂斯精英 样本中制备高通量蛋白质提取物和随后的共免疫沉淀的协议。

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

protein-extract-preparation-co-immunoprecipitation-from

Research

JoVE Journal

Developmental Biology

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

8.6K 次观看.  Kansas State University. 该方法描述了从 卡诺哈布迪蒂斯精英 样本中制备高通量蛋白质提取物和随后的共免疫沉淀的协议。

视频: Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

线虫中耳炎群链球菌引起氧化应激的研究

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

线虫生殖道内精子引导和运动的测定

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...