作者非常感谢由美国国立卫生研究院P50GM103297，P30CA100730和综合癌症P01CA16058支持。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 缓冲成分 </td></tr><tr><td> 洗涤液: </td></tr><tr><td>的50mM的Tris-HCl，pH 7.4的</td></tr><tr><td> 150毫米氯化钠</td></tr><tr><td> 3毫米氯化镁</td></tr><tr><td> 低盐缓冲液: </td></tr><tr><td>的20mM的Tris-HCl，pH值7.5 </td></tr><tr><td> 10毫米氯化钠</td></tr><tr><td> 3毫米氯化镁</td></tr><tr><td> 2毫摩尔DTT </td></tr><tr><td> 1×蛋白酶抑制剂鸡尾酒无EDTA </td></tr><tr><td>核糖核酸酶出5微升/毫升（RNA酶抑制剂） </td></tr><tr><td> 细胞质裂解液: </td></tr><tr><td> 0.2M的蔗糖</td></tr><tr><td> 1.2％的Triton X-100 </td></tr><tr><td> NETN-150洗涤液: </td></tr><tr><td>的20mM的Tris-HCl，pH 7.4的</td></tr><tr><td> 150毫米氯化钠</td></tr><tr><td> 0.5％的NP-40 </td></tr><tr><td> 3毫米氯化镁</td></tr><tr><td> 10％甘油</td></tr><tr><td> 结合缓冲液: </td></tr><tr><td>的10mM HEPES pH 7.6的</td></tr><tr><td> 40毫米氯化钾</td></tr><tr><td> 3毫米氯化镁</td></tr><tr><td> 2毫摩尔DTT </td></tr><tr><td> 5％甘油</td></tr></tbody></table> 表2:缓冲组合物。 1.细胞及亲和基质的制备<ol><li>培养的兴趣在10cm皿分汇合（80％）的细胞系。使用了FLAG标签NLS-MS2外壳蛋白的免疫沉淀无关（IP）10厘米板。执行使用抗血清的FLAG表位标签的IP。当表达FLAG标签的MS2外壳蛋白质粒转染细胞24-48小时提前收获20。 注:特定的RNP可以从核或细胞质中富集集成电路裂解物或生物化学分级分离制剂，如蔗糖梯度的级分。建议收获来自非转染细胞的裂解物在平行于提起附加阴性对照。 </li><li>每转的G蛋白磁珠浆IP 60微升至1.7 ml离心管。 </li><li>放置在磁铁架的管离心管到珠子从存储溶液中分离出来。 </li><li>用微量精心绘制，将其取出存储解决方案。 </li><li>从磁体机架上卸下管。 </li><li>洗涤和平衡1×洗涤缓冲液600微升珠（珠的使用量的10倍）（20毫摩尔Tris盐酸，pH为7.4; 3毫MgCl 2的;以及150mM的氯化钠）和最终过端3旋转分钟，在室温。 </li><li>放置在磁铁架管和除去洗涤缓冲液。 </li><li> 1×洗涤缓冲液和免疫沉淀FLAG抗体的10体积（600微升）添加到平衡的公关oteinģ磁珠（根据由生产用于免疫沉淀推荐的量）为至少30分钟至缀合的免疫沉淀抗体和旋转终端过端在室温下。使用相应的同种型IgG作为一个合适的抗体的阴性对照。 </li><li>放置在磁铁架的管收集珠子并除去上清液。 </li><li>从磁体机架上卸下管中，用1×洗涤缓冲液和旋转的10体积（600微升）洗抗体缀合的小珠在室温下3分钟。重复此步骤两次。 </li><li>放置在磁铁架管和除去洗涤缓冲液。 </li></ol> 2.收获的RNP 注:在步骤1.8之后的孵化时间准备的RNP。 <ol><li>通过抽吸除去从细胞培养基中并用1-5毫升冰冷的1X磷酸盐缓冲盐水（PBS）洗细胞两次。使用细胞刮赶走潮湿，在4℃收集前贴壁细胞通过在226×g离心4分钟离心。 </li><li>添加375微升冰冷却的，低盐缓冲液（20mM的Tris-HCl，pH值7.5; 3毫MgCl 2的10毫摩尔NaCl; 2mM的DTT; 1×蛋白酶抑制剂鸡尾酒，无EDTA;和5μl/ ml的RNA酶抑制剂）的细胞沉淀，并允许通过将它放置在冰上5分钟肿胀。 注:裁缝根据细胞沉淀的大小低盐缓冲液体积。对于从10cm平板1.2×10 6个细胞，375微升缓冲液中是足够的。 </li><li>收集胞质细胞裂解物中，添加125微升冰冷的裂解缓冲液（0.2M蔗糖/ 1.2％的Triton X-100），然后执行10笔用Dounce匀浆，这是在一个冰桶预冷。 注意:为了收集总细胞提取物，被推荐用于增溶的核质/染色质标准的RIPA裂解缓冲液。 </li><li>自旋以最快的速度（16,000 XG）1分钟微量;这将清除碎片的上清液第二核。 </li><li>将上清转移到新的1.7 ml离心管，其已经在冰上。确定由一个标准，实验室优选的方法，如Bradford测定23的总蛋白浓度。保留至少10％的等分试样用于蛋白质印迹分析，以用作一个输入控制。立即使用收获的细胞裂解物进行免疫沉淀或储存在-80℃以供将来分析。 注:根据我们的经验，这些样品可以存储和几个月后用于免疫分析没有诚信的一种妥协。 </li></ol> 3.免疫沉淀<ol><li>添加收获细胞裂解物的所需体积，基于在步骤2.5中确定的蛋白浓度，对目标抗体缀合的小珠。用1×洗涤缓冲液，使总体积达600微升和旋转终端过端在室温下90分钟。 注:该时间周期足以产生高亲和性的RNP复合体的健壮隔离，同时最小化非特异性结合。稀替代RNP来源，如蔗糖梯度的级分，至少1:1与1×洗涤缓冲液，然后用预先制备的珠 - 抗体复合孵育它们，如上所述。 </li><li> 90分钟温育后，放置在磁铁架知识产权管收集珠子。保留上清液作为流通。 注:此第一流通是衡量IP效率的重要对照样品。免疫印迹测定法将提供的IP效率的指示，以及所研究的相互作用的特异性。它建议保留该上清液用于下游分析。 </li><li> 150毫摩尔NaCl; 3毫MgCl 2的0.5％NP40;用冰冷的NETN-150洗涤缓冲液（20mM的Tris-HCl，pH值7.4的10体积（600微升）洗RNP结合，抗体缀合的小珠和10％甘油）根据在室温下3分钟的旋转。重复步骤3次。 注:此缓冲区从裂解缓冲液的不同，有效地降低了弱的或非特异性的关联。 </li><li>以下最后的洗涤，悬浮固定化的RNP复合物在60微升冰冷的1X结合缓冲液（10mM HEPES，pH值7.6; 40毫氯化钾; 3毫米氯化镁2; 5％甘油;和2mM DTT）中，并储备10％对于Western印迹和RNA分离20％的固定的复合物珠粒。 </li></ol> 4.洗脱<ol><li>调整剩余体积至100μl用冰冷的1X结合缓冲液，并在70℃下加热管3分钟。 </li><li>以分离同源RNA-蛋白质复合物，添加〜30-nt的DNA寡核苷酸是与感兴趣的RNA的3&#39;序列的边界互补并在室温下在温和温育30分钟摇动（100nM的寡核苷酸是足够了）。 注:寡核苷酸是反义邻近于翻译的核苷酸残基开始现场用作为例四氯乙烯构建体E在此协议。适当的序列互补性是由〜40％的GC含量提供。设计用于高效杂交与靶RNA的最小互补区域的反义寡核苷酸。 </li><li>添加5-10单位RNA酶H的该管，在室温下孵育1小时以裂解RNA的RNA:DNA杂交体。将上清转移到无菌1.7毫升离心管中;该样品中含有的利益所捕获的RNP复合物。 注:根据同源RNA的可获得性，两个或更多个轮RNA酶H处理可能是有用的，以增加样品丰度为下游分析。 </li><li>用洗脱液为20％的Western印迹已知相关蛋白并随后通过RT qPCR的洗脱液用于RNA分离的20％。剩下的60％可以用于质谱或鉴定蛋白质组分。 </li><li>平行地，若使保留细胞裂解物对RNA分离和RT-PCR分析的等分试样。这ASSEssment提供RNP园区内的RNA富集的迹象。 注:使用在控制免疫印迹分离蛋白制剂，以确定该核糖核酸酶H裂解能有效地从immunuoprecipitated复杂释放RNP。 </li></ol> 5.蛋白电泳和Western blot分析<ol><li>根据标准的实验室协议受试者的总样品进行SDS-PAGE和免疫印迹分析的约10-20％。 注意:此步骤用于之前下游RNA分析验证的IP效率和特异性。它也可以用来评估所述分离的RNP复合体的蛋白质组合物。 </li></ol> 6.收集免疫沉淀的RNA 注:RNA分离可通过Trizol法或以下所描述的协议来实现。 <ol><li>在750微升异硫氰酸胍试剂样品总数的一半悬浮和孵化在房T5分钟提取从捕获的PCE-RNP复合物的RNA之前emperature。 </li><li>加入200μl的氯仿至管，剧烈振荡10秒，并在室温下孵育3分钟。 </li><li>在4℃下16000×g离心15分钟离心后，收集水相到重新管和添加异丙醇等体积。拌匀并在室温下孵育至少10分钟。加入1μl乙二醇蓝色的样品，并在冷冻高效沉淀或加工-20℃存放在未来某一日期。 </li><li>离心反应管以16,000 xg离心在4℃下10分钟。小心收集并弃去上清液，以便不干扰RNA沉淀;建议使用的p-200微管尖端，以促进该过程。 </li><li> 16,000 xg离心加入500μl75％乙醇到每个管中，涡旋，离心管5分钟，在4℃下。认真收集并弃去上清液按步骤6.4。空气-干燥在100μl的无RNase水2-3分钟，重悬沉淀。 注意:不要延长时间沉淀晾晒，以避免与悬浮的一个问题。 </li><li>应用100微升RNA样品的RNA清理柱。使用制造商的协议处理。洗脱在无RNase水和存储的30微升的RNA在-80℃长达3个月。 注意:此分离的RNA适合于通过RT-实时PCR（qPCR的），微阵列，和RNA测序下游分析。取决于丰RNP和与感兴趣的RNP分离效率，相同的裂解物可以经受两个或更多轮的IP，以产生适用于下游分析足够的RNA。 </li></ol> 7. RNA反转录和扩增的cDNA通过PCR <ol><li>若使分离的RNA通过用高质量的逆转录酶（RT）的随机引物反转录，根据厂家专业cturer的说明。 </li><li>为了扩增目的RNA，设计核糖核酸酶H裂解序列内的特定基因的反义引物。使用反义寡核苷酸，随机引物或寡-dT引物相似量的（见材料表 ）。 注:寡聚-dT引物和聚腺苷酸化的mRNA提供用于RT-PCR反应阳性对照反应。 </li><li>储备RT反应（1微升）用于制备的qPCR串联做阴性对照溶胞产物的IP和阳性对照DNA样本来定义截止和产生的标准曲线的5％。如果制备的qPCR的CT值是超出标准曲线的范围内，稀释在1个连续的RT反应:5稀释液，并重复步骤7.2。 注:对于RNA测序和质谱技术，请参阅补充方法文件。 <a href="http://ecsource.jove.com/files/ftp_upload/54391/Supplementary_method_File_Jove.docx" target="_blank">请点击此处Ť</a>Ø查看本补充方法文件。 （右键点击下载）。 </li></ol>

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Virol. 73 (6), 4847-4855 (1999).</li><li>Bolinger, C., 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=RNA+helicase+A+interacts+with+divergent+lymphotropic+retroviruses+and+promotes+translation+of+human+T-cell+leukemia+virus+type+1.">RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1.</a> Nucleic Acids Res. 35 (8), 2629-2642 (2007).</li><li>Bolinger, C., Sharma, A., Singh, D., Yu, L., Boris-Lawrie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+helicase+A+modulates+translation+of+HIV-1+and+infectivity+of+progeny+virions.">RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions.</a> Nucleic Acids Res. 38 (5), 1686-1696 (2010).</li><li>Lee, T., 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=Suppression+of+the+DHX9+helicase+induces+premature+senescence+in+human+diploid+fibroblasts+in+a+p53-dependent+manner.">Suppression of the DHX9 helicase induces premature senescence in human diploid fibroblasts in a p53-dependent manner.</a> J.Biol.Chem. 289 (33), 22798-22814 (2014).</li><li>Bradford, M. 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+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal.Biochem. 72, 248-254 (1976).</li><li>Glenn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+protein+samples+for+mass+spectrometry+and+N-terminal+sequencing.">Preparation of protein samples for mass spectrometry and N-terminal sequencing.</a> Methods Enzymol. 536, 27-44 (2014).</li><li>Keene, J. D., Komisarow, J. M., Friedersdorf, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIP-Chip:+the+isolation+and+identification+of+mRNAs,+microRNAs+and+protein+components+of+ribonucleoprotein+complexes+from+cell+extracts.">RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts.</a> Nat.Protoc. 1 (1), 302-307 (2006).</li><li>Ranji, A., Shkriabai, N., Kvaratskhelia, M., Musier-Forsyth, K., Boris-Lawrie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+double-stranded+RNA-binding+domains+of+RNA+helicase+A+are+necessary+for+selective+recognition+and+translation+of+complex+mRNAs.">Features of double-stranded RNA-binding domains of RNA helicase A are necessary for selective recognition and translation of complex mRNAs.</a> J.Biol.Chem. 286 (7), 5328-5337 (2011).</li></ol>

The authors have nothing to disclose.

这里描述的RNP隔离和同源RNA识别策略是调查一个特定的RNA蛋白质相互作用，发现候选蛋白共同调节细胞的特异性RNP的选择性手段。 使用寡核苷酸定向RNA酶H切割以分离的RNP的优点是捕捉和具体分析过下游结合到感兴趣的顺式作用RNA元件异质的RNP的RNP顺式作用RNA元件的能力。因为在给定的RNP同源RNA的丰度是无RNA酶H裂解分离所收集的RNP的一小部分，该工作流的主要缺点是同源的RNP...

转录后基因表达被精确调节，以在细胞核中的DNA的转录开始。通过RNA结合蛋白（限制性商业惯例）控制，基因生物合成及代谢发生在高度动态的核糖核蛋白颗粒（体RNP），其联营公司及RNA代谢1-3的进展过程中与底物前体mRNA的解离。在RNP部件的动态变化影响的mRNA的转录后命运和初级成绩单，它们的核运输和定位，它们作为mRNA翻译模板活动，和成熟的mRNA的最终成交的处理过程中提供质量保证。 许多蛋白质由于其保守的氨基酸结构域，包括RNA识别基序（RRM）的指定为限制性商业惯例中，双链RNA结合结构域（RBD），和碱性残基的延伸（ 例如，精氨酸，赖氨酸，和甘氨酸） 4。限制性商业惯例通常是通过免疫策略分离和筛选，以确定其同源的RNA。某些限制性商业惯例共调节预的mRNA即在功能上相关的，被指定为RNA的调节子5-8。这些限制性商业惯例，其同源的mRNA，有时非编码RNA，形成催化的RNP在组成而改变;其独特之处是由于相关因素的各种组合，以及以时间顺序，位置，以及它们之间的相互作用9的持续时间。 RNA免疫沉淀（RIP）是一个强大的技术来隔离细胞的RNP，并确定使用序列分析10-13相关的成绩单。从候选移动到全基因组筛选是通过RIP与微阵列分析14或高通量测序（RNA测序）15组合是可行的。同样地，共沉淀的蛋白质可通过质谱法鉴定，如果它们足够丰富和可分离来自共沉淀的抗体16，17。在这里，我们解决了分离从培养的人类细胞的特定的同源RNA的RNP部件的方法，虽然该方法是可改变的植物细胞，真菌，病毒和细菌的可溶性裂解物。该材料的下游分析包括候选识别和验证通过免疫印迹，质谱法，生化酶促测定法，RT-qPCR的，微阵列，和RNA测序，如总结于图1。 定的RNP在在转录后水平控制基因表达的基本作用，在组分限制性商业惯例或它们的可访问的表达的改变，以同源的RNA可能是有害的细胞，并与几种类型的疾病，包括神经系统疾病18相关联。 DHX9 / RNA解旋酶A（RHA）是必要的细胞和逆转录病毒起源6选定的mRNA的翻译。这些同源的RNA具有内结构相关的顺式作用元件的5&#39;端非编码区，其被指定为转录后控制元件（PCE）19。 RHA-PCE活性是必需的许多逆转录病毒，包括HIV-1的有效帽依赖性翻译，和生长调节基因，包括JUND 6,20,21。由必需基因（dhx9）编码，RHA是细胞增殖和其下调基本消除了细胞活力22。 RHA-PCE的RNP的分子分析对于理解为什么RHA-PCE活性是必要的，以控制细胞增殖的重要步骤。 在稳定状态下或在细胞的生理扰动RHA-PCE RNP组分的确切性质要求RHA-PCE的RNP在足够丰富用于下游分析选择性富集和捕获。这里，逆转录病毒PCEgag的RNA具有标记为开放阅读框架内的MS2外壳蛋白（CP）的顺式作用的RNA结合位点的6份。该MS2外壳蛋白外切genously共表达质粒转染PCEgag的RNA以促进生长的细胞的RNP组件。含有同源MS2标记PCEgag RNA中的MS2外壳蛋白的RNP从细胞提取物中免疫沉淀和磁珠（ 图2a）捕获。选择性地捕获结合于四氯乙烯的RNP部件，固定RNP用至远端的PCE序列互补的寡核苷酸温育，形成的RNA-DNA杂交是对RNA酶H活性的底物。因为四氯乙烯是位于非翻译区的5&#39;5的终端"，寡核苷酸是相邻的逆转录病毒翻译起始位点（的gag起始密码子）的RNA序列互补。邻近在gag起始密码子从固定化的RNP，将其收集作为洗脱剂释放的5&#39;非编码区复杂RNA酶H裂解。此后，将样品通过RT-PCR评估，以证实PCEgag的，并通过SDS PAGE和免疫印迹捕获确认captu重新目标MS2外壳蛋白。四氯乙烯相关的RNA结合蛋白的验证，DHX9 / RNA解旋酶A，然后进行。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dynabeads Protein A</td>
			<td>Invitrogen</td>
			<td>10002D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td>10004D</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- FLAG antibody</td>
			<td>Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- FLAG antibody</td>
			<td>Sigma</td>
			<td>F7425</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-RHA antibody</td>
			<td>Vaxron</td>
			<td>PA-001</td>
			<td></td>
		</tr>
		<tr>
			<td>TRizol LS reagent</td>
			<td>Life technology</td>
			<td>10296-028</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase H</td>
			<td>Ambion</td>
			<td>AM2292</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26184</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaeasy clean-up column</td>
			<td>Qiagen</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>Omniscript reverse transcriptase</td>
			<td>Qiagen</td>
			<td>205113</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Out</td>
			<td>Invitrogen</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>98379</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>17904</td>
			<td></td>
		</tr>
		<tr>
			<td>Random hexamer primers</td>
			<td>Invitrogen</td>
			<td>N8080127</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo-dT primers</td>
			<td>Invitrogen</td>
			<td>AM5730G</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>IDT</td>
			<td></td>
			<td>Gene specific primers for&#160; PCR amplification</td>
		</tr>
		<tr>
			<td>Oligonucleotide for RNase H mediated cleavage</td>
			<td>IDT</td>
			<td></td>
			<td>Anti-sense primer for target RNA</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco Life technology</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM tissue culture medium</td>
			<td>Gibco Life technology</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco Life technology</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S642-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP214</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>R0862</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Bio-Rad</td>
			<td>1620112</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td></td>
			<td></td>
			<td>1.7 ml micro-centrifuge tube holding</td>
		</tr>
		<tr>
			<td>Laminar hood</td>
			<td></td>
			<td></td>
			<td>For animal tissue culture</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td></td>
			<td></td>
			<td>For animal tissue culture</td>
		</tr>
		<tr>
			<td>Protein gel apparatus</td>
			<td></td>
			<td></td>
			<td>Protein sample separation</td>
		</tr>
		<tr>
			<td>Protein transfer apparatus</td>
			<td></td>
			<td></td>
			<td>Protein sample transfer</td>
		</tr>
		<tr>
			<td>Ready to use protein gels (4-15%)</td>
			<td></td>
			<td></td>
			<td>Protein sample separation</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td></td>
			<td></td>
			<td>Pellet down the sample</td>
		</tr>
		<tr>
			<td>Table top rotator</td>
			<td></td>
			<td></td>
			<td>Mix the sample end to end</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td>Mix the samples</td>
		</tr>
	</tbody>
</table>

isolation of cognate rna-protein complexes from cells using oligonucleotide-directed elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

RIP前鉴定结果逆转录病毒插科打诨RNA和选择的共沉淀的细胞的RNA与DHX9 / RHA，包括HIV-1 6，勇得6户珥（弗里茨和鲍里斯-劳列，未发表）。反转录病毒的5&#39;非编码区已被证明与DHX9 / RHA在细胞核共沉淀并在多核糖体的细胞质共同隔离。它是唯一定义为顺式短效后转录控制元件（PCE）6。为了分离形成增殖细胞PCEgag的RNA核糖核蛋白RHA复合物（体RNP...

Watch this Scientific Journal Video about Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution at JoVE.com

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

isolation-cognate-rna-protein-complexes-from-cells-using

Research

JoVE Journal

Biochemistry

8.9K 次观看.  University of Minnesota. This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A. 

视频: Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

使用非变性聚丙烯酰胺凝胶电泳人的MxA多亚基蛋白复合物的表征

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

科研

生物化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

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 ...

Rapid Isolation of the Mitoribosome from HEK Cells

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

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

HEK 细胞 Mitoribosome 的快速分离

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

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Isolation of Tissue Extracellular Vesicles from the Liver

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

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

组织细胞外囊泡与肝脏分离

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

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...