Name: protein extract preparation and co-immunoprecipitation from caenorhabditis elegans
Uploaded: 2020-05-23T15:00:00.000+00:00
Duration: 7 min 22 s
Description: Watch this Scientific Journal Video about Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans at JoVE.com

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>

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

8.8K 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

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

科研

癌症研究

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

生物化学

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

生物学

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

线虫的核苷酸和蛋白质提取联合

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

卡诺尔哈布炎细胞外囊泡的纯化与分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble proteins over 40% of the time results in an extension of the lifespan in C. elegans, suggesting that many of these proteins are key determinants of the aging process. Isolation and quantitative identification of these insoluble proteins are crucial to understand key biological processes that occur during aging. Here, we present a modified and improved protocol that details how to extract and isolate the SDS-insoluble proteins (insolublome) from C. elegans more efficiently to streamline mass spectrometric workflows via a novel label-free quantitative proteomics analysis. This improved protocol utilizes a highly efficient sonicator for worm lysis that greatly increases efficiency for protein extraction and allows us to use significantly less starting material (approximately 3,000 worms) than in previous protocols (typically using at least 40,000 worms). Subsequent quantitative proteomic analysis of the insolublome was performed using data-dependent acquisition (DDA) for protein discovery and identification and data-independent acquisition (DIA) for comprehensive and more accurate protein quantification. Bioinformatic analysis of quantified proteins provides potential candidates that can be easily followed up with other molecular methods in C. elegans. With this workflow, we routinely identify more than 1000 proteins and quantify more than 500 proteins. This new protocol enables efficient compound screening with C. elegans. Here, we validated and applied this improved protocol to wild-type C. elegans N2-Bristol strain and confirmed that aged day-10 N2 worms showed greater accumulation of the insolublome than day-2 young worms.

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

This novel workflow efficiently extracts and isolates SDS-insoluble proteins (insolublome) from Caenorhabditis elegans with minimal starting material for quantitative differential proteomic analysis. The protocol uses a comprehensive data-independent acquisition mass spectrometry analysis to quantify the insolublome and bioinformatic analysis to gain biological insights into aging mechanisms and pathologies.

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

小规模提取 秀丽隐杆线虫 基因组DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...

Single Cell Dissociation of Caenorhabditis elegans: A Method to Isolate Live Cells

Source: Germany, E. M., et al. <a href="https://www.jove.com/video/60145/isolation-specific-neuron-populations-from-roundworm-caenorhabditis">Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans</a>. J. Vis. Exp. (2019).
This video describes a method to dissociate whole worms into a single-cell suspension suitable for FACS or immunoprecipitation of intact cells.

秀丽隐杆线虫的单细胞解离:一种分离活细胞的方法

Source: Germany, E. M., et al. Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans. J. Vis. Exp. (2019).
This video describes a ...

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

摘要

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使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

活性秀丽隐杆线虫核提取物的分离及重建用于体外转录

前列腺素的提取与分析

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

线虫的核苷酸和蛋白质提取联合

卡诺尔哈布炎细胞外囊泡的纯化与分析

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

小规模提取秀丽隐杆线虫基因组DNA

秀丽隐杆线虫的单细胞解离:一种分离活细胞的方法

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

摘要

探索更多视频

此视频中的章节

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

前列腺素的提取与分析

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

线虫的核苷酸和蛋白质提取联合

卡诺尔哈布炎细胞外囊泡的纯化与分析

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

小规模提取 秀丽隐杆线虫 基因组DNA

秀丽隐杆线虫的单细胞解离:一种分离活细胞的方法

活性秀丽隐杆线虫核提取物的分离及重建用于体外转录

小规模提取秀丽隐杆线虫基因组DNA