国防部（创意奖W81XWH-07-0406） - 乌鲁斯阿塔索伊 NIH RO1 A1080870 - 乌鲁斯阿塔索伊 NIH R21 A1079341 - 乌鲁斯阿塔索伊密苏里州的机构资金 - 乌鲁斯阿塔索伊

实验准备在开始实验之前，这是关键的是具有所有试剂，容器和用具，无RNase。治疗玻璃器皿与RNase抑制剂（RNaseZAP，Ambion公司），然后用DEPC处理过的水冲洗。确保所有的试剂被确认为无RNase。 1。准备mRNP裂解液<ol><li>种植和收获成倍增长的组织细胞产生的总蛋白2-5毫克之间，每个RIP。 <ol class="lalpha"><li>两个P150培养皿通常就足够了。 </li><li>对于每个被调查的RBP，总细胞数和蛋白质的量必须进行优化，以最大限度地提高相应的靶mRNA和RBP的相互作用。 </li><li> RIP定量RT-PCR分析，可能需要较少的细胞裂解液（约400μg总蛋白），由于HuR的TIAR，AUF1，尤其是对高丰度的限制性商业惯例，其扩增和检测方法。 </li></ol> &lt;/ P&gt; <li>颗粒细胞通过离心200×g离心8-10分钟，在4°C，然后用冰冷的PBS洗三次。 </li><li>最后一次洗涤后，轻轻地滑动管的底部和重悬细胞沉淀，在制备的多核糖体RNase及蛋白酶抑制剂的裂解缓冲液（PLB）与等体积的。 <ol class="lalpha"><li>建议测量的准确体积的细胞沉淀。 </li></ol></li><li>轻轻地移液管混合（涡），掰开细胞团块，和裂解液在冰上孵育5分钟。 </li><li>离心机在13,000 xg离心20分钟，在4℃至清除杂物的溶胞产物。 </li><li>立即转移到预冷的微量离心管中清零上清液。 </li><li>查看在冰上并储存在-80℃下<ol class="lalpha"><li>立即冻结完成适当的裂解细胞，并防止不必要的绑定。继续与RIP紧随其后的样品融化和冰样品，以避免RNA的降解。/ P&gt; <li>此溶胞产物可以存储最多六个月在-80℃下应避免反复冻融循环，因为这可以导致蛋白质和/或mRNA的降解。 </li></ol></li><li>在使用标准布拉得福德蛋白质分析的溶胞产物，测定蛋白质浓度。 </li></ol> 2。外壳蛋白A琼脂糖珠与抗体和冲洗<ol><li>预膨胀蛋白A琼脂糖凝胶（PAS）珠过夜，用5％BSA在NT2缓冲（3-4卷）。存放于4℃ <ol class="lalpha"><li>长期贮存几个月在4°C是可能的时，补充有0.1％的叠氮化钠。 </li><li>目标RBP抗体同种型的基础上，应选择蛋白质Sepharose磁珠。蛋白A，G和A / G都有特定的抗体亚型目标和不同的目标亲和力。蛋白A琼脂糖珠的基础上同型HuR的蛋白抗体的特异性和亲和力。 </li></ol></li><li>前使用，去除多余的的NT2缓冲，所以最终珠缓冲区比为1:1。 </li><li>用1.5ml无RNA酶的离心管中，取出100微升的PAS浆，加入30微克的抗体反应（ 即 ，具体RBP及同型对照）每个IP。 </li><li>加入100-200微升NT2缓冲抗体珠组合。 <ol class="lalpha"><li>混合物可被存储为几个星期，在4℃时，补充有0.1％的叠氮化钠。 </li><li>同种型匹配的抗体或来自同一物种的整个正常血清应并行地使用在背景RNA作为抗体，该抗体控制。 </li></ol></li><li>珠混合添加相应的抗体，孵育过夜，比上年末翻滚在4°C。 <ol class="lalpha"><li>优化被调查的特定蛋白的抗体效价（1，5，10或30μg的抗体通常是足够的）。 </li></ol></li><li>准备使用前立即用1ml的洗涤的抗体包被的珠子冰冷却NT2缓冲器5倍。 <ol class="lalpha"><li>通过离心1-2分钟，在4℃下以13,000 xg离心珠混合洗净</li><li>小心地取下的最高金额supernantant手动移液器或吸气，但要小心，以避免干扰颗粒。 </li><li>洗衣机有助于除去未结合的抗体以及抗体混合物的RNA酶污染物。 </li></ol></li><li>一旦已经完成最后一次洗涤后，重悬在700微升冰冷的NT2缓冲区后跟治疗与各种RNase抑制剂的保护的靶mRNA，包括10微升的RNaseA暂在40单位/微升，10微升的100mM珠DTT和15μlEDTA（15毫米）。把音量到1000微升NT2缓冲。 <ol class="lalpha"><li>氧钒核糖核苷复合物由于不使用EDTA的抑制作用。 </li></ol></li></ol> 3。免疫沉淀和RNA沉淀<ol><li>可选的预清除步骤：<ol class="lalpha"><li>以减少背景，珠子和对照抗体，也可以使用预先明确的溶胞产物。这可能会降低信号在输出中。 </li><li>这一步可能需要减少的背景时，IP其次是芯片。它通常是没有必要的IP定量RT-PCR。 </li><li>预清除15微克的同型对照为30分钟/ 4°C比上年末翻滚。 </li><li>加入50μlpre​​swollen PAS珠涂有抗体的步骤2.1。 </li><li>孵育30分钟，比上年末/ 4°C旋转结束</li><li>在4℃下以10,000 xg离心保存上清IP。 </li></ol></li><li>加入100μl的隔离澄清的裂解液（约2-5毫克）制备的抗体组合。 <ol class="lalpha"><li>稀释裂解，将有助于减少背景和非特异性结合。 </li><li>裂解液输入量可能会有所不同，具体取决于检测方法和丰富的RBPØŕ效率RBP抗体所指出的步骤1.1.c. </li></ol></li><li> （可选）立即混合管轻轻轻弹数次，然后短暂离心，以10,000 xg于4℃下沉淀珠，并立即取出100μl的上清液作为输入总mRNA的表示qPCR分析使用标准的RNA分离技术。 <ol class="lalpha"><li>这一步是确认输入裂解RNA是最佳的IP，应该只执行一个检查步骤或差的RNA结果RIP后的故障排除步骤。 </li></ol></li><li>换行管封口膜，以确保密封和在4°C孵育2〜4小时，比上年末翻滚。 <ol class="lalpha"><li>优化的基础上孵化的时间应该在目标丰度，应尽量避免复杂的重排或退化。对于某些限制性商业惯例较短孵育可能会更理想。 </li></ol></li><li>颗粒珠在5000 XGF或5分钟，在4°C，保存上清潜力分析Western blot检测。将等分试样储存于-80℃。 <ol class="lalpha"><li>残余靶蛋白具有高量的等分的上清液可以指示故障的蛋白质沉淀琼脂糖珠。 </li></ol></li><li>如前所述，洗珠5次用1ml冰冷NT2缓冲液和离心（5,000×g离心5分钟，在4℃），然后去除上清液与手移液器或一个吸气。 <ol class="lalpha"><li>更严格的洗涤方法，可以使用以降低背景补充NT2脱氧胆酸钠缓冲液，尿素或SDS。 </li><li>样本保持尽可能多的冰和快速工作，以减少降解靶mRNA。 </li></ol></li><li>可选：一个小试样（10％），珠混合并行运行更多的样品检查IP效率的目标蛋白，用Western blot分析。</li></ol> 4。 DNA酶和蛋白酶K的治疗<ol><li>洗涤后，用100μl的NT2缓冲液重悬珠补充用5μl的无RNA酶的DNA酶1（2 U /微升）。 </li><li>请在37℃下5-10分钟。加入1毫升的NT2缓冲区，并在5,000 xg离心1分钟，在室温下离心。 <ol class="lalpha"><li>可以使用一个小的（〜10％）的等分试样的珠子来检查IP通过SDS-PAGE分析的效率。 </li></ol></li><li>首席助理局长沉淀重悬在100μlNT2缓冲液，5微升的蛋白酶K（10毫克/毫升），和1μl10％SDS。 </li><li>孵育30分钟，再悬浮的胎圈混合物在55℃下在水浴中，在每10分钟轻轻轻弹。 <ol class="lalpha"><li>蛋白酶K，将有助于在释放的RNP组件。 </li></ol></li><li>佩莱珠在5,000 xg离心5分钟，在室温（RT），并收集上清液（约100微升）。 </li><li>加入200μlNT2缓冲珠，离心华夫格5000×g离心2分钟，在室温下，收集上清液（约200μL），并丢弃珠。 </li><li>合并上清液（加入100μl和200μl），并添加300微升的酸苯酚-CHCl 3下层。 </li><li>涡，1分钟室温（或37℃下在摇动器）和在RT下以16,000 xg离心1分钟的离心分离机。 </li><li>收集250微升的上层（不破坏接口），加入25μL醋酸钠在pH值5.2，625μL100％的乙醇和5μlglycoblue，拌匀。 </li><li>贮存于-20℃下过夜<ol class="lalpha"><li>的RNA，以确保适当的恢复，此外glycoblue增加的可见性通过作为载体分子的RNA沉淀。 </li></ol></li><li>翌日，混合管由反演的3-5倍，自旋以12,000 xg于4℃下30分钟，并弃去上清液。 </li><li>加入1毫升70％ETOH蓝色的颗粒和通过反转或涡旋混合。 </li><li>离心机在4℃下12,000 xg离心2分钟。 </li><li> ðiscard上清液和12,000 xg下离心1分钟，在4℃。 </li><li>除去任何残余的70％ETOH于RT的移液管和空气干燥沉淀5分钟。 </li><li>重悬在20〜40μl的RNA酶/无DNA酶水或其他适当的音量。 <ol class="lalpha"><li>现在已准备好进一步向下游应用，如实时定量RT-PCR或芯片的样品。 </li><li>的NanoDrop分光光度法可用于评估样品的浓度，然而，贵金属的样品中，它可能是有利的避免nanodropping样品，因为它可以是浪费和相对不准确的。我们建议正常化样本，以看家基因转录宝石或低收益率样本的纯度和IP效率评估。 </li></ol></li></ol> 5。代表性的成果如果程序进行了优化，并进行正确的免疫沉淀产生显着富集的mRNA靶标。通常情况下，根据RBP和它的靶mRNA（S），我们可以看到进行评估时，通过实时定量RT-PCR富集约10 - 50倍。许多限制性商业惯例的目标可以发现集体使用基因芯片分析。然而，这种方法是用实时定量RT-PCR方法相比更敏感的降解。根据上RBP，目标的数量和反应效率，微阵列可以揭示数百个新的目标，或者它可以只发现了很少，如果有的。例如，一个更好的特点RNA结合蛋白HuR的，转录后调控许多重要的生理基因1，2的表达和翻译。通过RIP芯片在乳腺癌细胞株中的HuR的ribonucleo复杂的分离，例如，显示的几个重要的已知HuR的目标，包括使用定量RT-PCR方法，如在图1中所示的β-肌动蛋白的富集。在这两种肿瘤细胞株的β-肌动蛋白富集的12 - 至15 - 倍。通常情况下，正确执行时，我们看到了显着的ENRichment各种细胞系中的β-肌动蛋白。然而，如果RIP没有发现任何显着的β-肌动蛋白的富集这表明与RIP的一个问题，可能需要重复该过程。此外，从这些细胞系的免疫沉淀的样品的微阵列分析的子集显示不同的表达HuR的目标在不同的雌激素受体 ​​（ER）阳性的MCF-7癌细胞与ER阴性MB-231乳腺癌细胞株作为相比，表明在图2 1。这些目标分为几类：已知和未知的HuR的目标，无论是与癌症有关或无关的。例如，CALM2和CD9都是以前没有HuR的目标确定为癌基因。使用微阵列和确认用定量RT-PCR，CALM2和CD9被发现是5 - 至180倍富集HuR的粒料中的指示突出HuR的蛋白质和这些目标基因之间的相互作用。 <i毫克SRC =“所有/ files/ftp_upload/3851/3851fig1.jpg”ALT =“图1”/&gt; 图1。免疫沉淀和RIP MB-231（ER）和MCF-7（ER +）乳腺癌细胞 MB-231，MCF-7细胞裂解液进行免疫沉淀。使用抗HuR的单克隆抗体（3A2）和IgG1同型对照。 A. IP西户珥透露，预计大小的带检测3A2。在右边的面板上显示金额，户珥的输入裂解液使用这两个细胞系，与β-微管蛋白作为加载控制。 B.定量RT-PCR验证表明，3A2 IP地址从MB-231和MCF-7，15和11倍的富集β-肌动蛋白，已知的HuR的目标。 ΔΔCT值进行标准化，以GAPDH。进行了实验，一式两份，每份（n = 2时）。 <img src="/files/ftp_upload/3851/3851fig2.jpg" alt="图2" /> 图2。 HuR的RIP-CHIP确定不同的基因图谱，在ER +和ER-的乳腺癌细胞。HuR的免疫沉淀进行从MB-231或MCF-7细胞的裂解物使用的HuR抗体和IgG1同种型对照的Illumina公司Sentrix阵列（47,000个基因）杂交。控制信号中减去。结果代表来自12个不同的阵列的累计数据。实验进行了每种细胞系相匹配的控制组（n = 3），一式三份。秤是LOG2。

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+Regulatory+Mapping+Indicates+that+the+RNA-Binding+Protein+HuR+Couples+Pre-mRNA+Processing+and+mRNA+Stability.">Integrative Regulatory Mapping Indicates that the RNA-Binding Protein HuR Couples Pre-mRNA Processing and mRNA Stability.</a> Molecular Cell. 43, 1-13 (2011).</li></ol>

作者宣称，他们有没有利益冲突。

由于这个实验的性质，优化和经验的唯一可靠的方法，成功地获得预期的结果。在此过程中的许多步骤，温度和所用试剂和产品的高效的处理是极为重要的。适当的规划和执行的技术将有助于确保该实验是在一个合理的时间在推荐的最佳温度。 RNA分离实验的一个主要问题是由核糖核酸酶的敏感性RNA的降解。所有的试剂需要无RNA酶的无RNase的容器中并贮存或使用。这是一个关键的步骤，以确保你的基因样本的完整性。甚至当实验正确执行，然而，期望的结果可能无法实现由于RBP和其靶mRNA之间的相互作用的性质。 一个潜在的问题是有低，甚至无信号提取的RNA RIP-芯片。虽然有可能是从总RNA中的信号，这可能是由珠子被拉下的结合蛋白不足的结果。第一个故障排除步骤是确认的细胞裂解液正在使用的具体RBP有足够的表达。经确认后，蛋白质可被分离后最终NT2洗涤并重新悬浮在Laemmli缓冲或另一种合适的变性缓冲液，并加热，在95℃的5分钟。 Western blot分析可以用在这些样品中的协调与输入裂解液作为阴性对照，以确保有足够的下拉相关蛋白。 此外，因为裂解细胞需要访问这些组件，通常分离的蛋白质和mRNA的异常和不必要的相互作用的潜在可能被引入。这些相互作​​用可能与“吸收”靶mRNA结合蛋白通过非特异性相互作用。此外，蛋白质在这些不同的合作条件可以折叠的多种变体，其结合基序可能变得不可访问他们的目标基因，防止它们之间的相互作用。这些都加强了工作效率，以及利用最佳温度限制这些有害的相互作用的重要性。此外，为每个特定的目标蛋白的洗涤条件的优化将是至关重要的最大化的相互作用的纯度。可能需要更严格的洗涤条件。例如，洗涤缓冲液可以被补充与SDS或一个适当的量的尿素，以减少非特异性的相互作用和背景中的信号输出。这将是完全依赖于实验者的目标的RBP以及作为其独特的生理条件下与靶mRNA中。在某些情况下，将不适合于某些mRNA的分析工具，应当指出，在制备的样品。 最后，虽然在富集的RNA RIP是成功-RBP的互动与RIP（CHIP）的方法，一个众所周知的问题是无法确定具体的RBP结合域的瞬时表达目标。几种交联技术可用于随后RIP隔离独特的序列目标，但是，使用短波UV趋于导致核酸损伤。一种新方法被称为的PAR-CLIP，或光活化的核糖核苷的交联剂和immuoprecipitation，采用长波UV纳入thiouridine成新生RNA允许从稳定和瞬时RNA的相互作用的独特的结合位点的识别。 总体而言，RIP-芯片已经建立了一个很好的工具，用来分离和研究组，以及许多其他的研究小组之间的相互作用RNA结合蛋白和mRNA的目标。虽然敏感的性质和实践，正确执行此过程会产生这些RNP复合物隔离，直到最近，已经INACC的发现和分析essible为。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 1 M二硫苏糖醇</td><td>费舍尔</td><td> BP172-5 </td><td> DTT </td></tr><tr><td> DNA酶1 </td><td> Ambion公司</td><td> 2235 </td><td>无RNase </td></tr><tr><td>乙二胺Tetraccetic酸</td><td>费舍尔</td><td> BP118-500 </td><td> EDTA </td></tr><tr><td>糖原</td><td> Ambion公司</td><td> 9516 </td><td></td></tr><tr><td> 1 HEPES </td><td>西格玛</td><td> H3375-100G </td><td> pH值7.0 </td></tr><tr><td>的Igepal NONIDET P-40 </td><td> USB </td><td> 78641 </td><td> NP40 </td></tr><tr><td> 1 M氯化钾</td><td>费舍尔</td><td> BP366-500 </td><td></td></tr><tr><td> 1 M的MgCl 2 </td><td>费舍尔</td><td>乙P214-500 </td><td></td></tr><tr><td> NT2缓冲区</td><td>见下面</td><td></td><td></td></tr><tr><td>多聚核糖体裂解缓冲液</td><td>见下面</td><td></td><td></td></tr><tr><td>蛋白酶抑制剂鸡尾酒粒的</td><td>罗氏公司</td><td> 11873580001 </td><td></td></tr><tr><td>蛋白A Sepharose磁珠</td><td>西格玛</td><td> P3391 </td><td></td></tr><tr><td>蛋白酶K </td><td>费舍尔</td><td> BP1700-100 </td><td></td></tr><tr><td>核糖核酸酶出核糖核酸酶抑制剂</td><td> Invitrogen公司</td><td> 10777-019 </td><td> 40 U /μL </td></tr><tr><td> 1M NaCl的</td><td>费舍尔</td><td> BP358-212 </td><td></td></tr><tr><td>十二烷基硫酸钠</td><td>费舍尔</td><td> BP166-500 </td><td> SDS </td></tr><tr><td> 1 M的Tris-HCl </td><td>费舍尔</td><td&gt; BP153-500 </td><td> pH值7.4 </td></tr><tr><td> Trizol法</td><td> Invitrogen公司</td><td> 15596-026 </td><td></td></tr><tr><td>钒核糖核苷复合物</td><td>新英格兰实验室</td><td> S1402S </td><td> VRC </td></tr></tbody></table> <table border="1"><tbody><tr><td> 试剂全面评估 </td></tr><tr><td>核糖核酸酶/无DNase的，DEPC处理过的玻璃器皿中制备试剂</td></tr><tr><td> 多聚核糖体裂解缓冲液 </td></tr><tr><td> 100毫米氯化钾</td></tr><tr><td> 5毫摩尔MgCl 2 </td></tr><tr><td>的10mM HEPES（pH7.0）中</td></tr><tr><td> 0.5％NP40 </td></tr><tr><td> 1毫米DTT </td></tr><tr><td> 100单位/毫升核糖核酸输出</td></tr><tr><td> 400μMVRC </td></tr><tr><td>蛋白酶抑制剂鸡尾酒平板的</td></tr><tr><td> 5毫升的多核糖体的裂解缓冲液 </td></tr><tr><td>添加50μl的1 M HEPES（pH值7.0） </td></tr><tr><td> 500μl的1 M KCL </td></tr><tr><td> 25微升的1 M的MgCl 2 </td></tr><tr><td> 25μl的NP40 </td></tr><tr><td> 4.7毫升无RNA酶的DNA酶H 2 O </td></tr><tr><td> 50微升的1 M DTT </td></tr><tr><td> 100 U / ml的RNA酶12.5μl的出</td></tr><tr><td> 200μl蛋白酶抑制剂混合物（溶于根据制造商） </td></tr><tr><td> 10微升的200mM VRC（在使用时） </td></tr><tr><td> NT2缓冲区 </td></tr><tr><td>的50mM的Tris-HCl（pH7.4）中</td></tr><tr><td> 150mM NaCl的</td></tr><tr><td> 1毫摩尔MgCl 2 </td></tr><tr><td> 0.05％NP40 </td></tr><tr><td> 1 L的NT2缓冲器 </td></tr><tr><td>的50毫升Tris（pH7.4）中</td></tr><tr><td> 30毫升5 M氯化钠</td></tr><tr><td> 1毫升1 M氯化镁<suB&gt; 2 </td></tr><tr><td> 500μLNP40 </td></tr><tr><td> 820毫升RNA酶，无DNA酶H 2 O </td></tr></tbody></table>

method for the isolation and identification of mrnas, micrornas and protein components of ribonucleoprotein complexes from cell extracts using rip-chip

作为发展的高通量测序和高效的微阵列分析的结果，全球基因表达分析已经成为一个简单的数据收集和随时可用的形式。在许多研究，然而，疾病模型，靶基因mRNA的稳态水平不总是直接与稳态蛋白水平。基因转录后调控是一个可能的解释两者之间的分歧。结合的RNA结合蛋白（RBP）的带动下，形成核糖核蛋白（RNP）复合物与靶mRNA的转录后调控影响mRNA的定位，稳定性和翻译的。确定这些未知从头目标mRNA的细胞提取物中的RNP复合物的RBP和其造成的影响对蛋白质的输出理解的机制和功能的关键。该协议概述的方法被称为RNP免疫沉淀芯片（RIP-Chip）的，允许s的识别pecific的mRNA的核糖核蛋白复合物，在试验条件下，随着选项，以进一步优化的个别研究者的实验。有了这个重要的实验工具，研究人员能够探索与转录后基因调控，以及其他核蛋白的相互作用的复杂机制。

Watch this Scientific Journal Video about Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip at JoV

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

一步一步协议通过RIP-芯片的分离和鉴定RNA相关的复合物。

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

method-for-isolation-identification-mrnas-micrornas-protein

Research

JoVE Journal

Biology

27.4K 次观看.  University of Missouri. 一步一步协议通过RIP-芯片的分离和鉴定RNA相关的复合物。

视频: Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

科研

生物学

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

过程评价絮凝剂的效率的分散粒子的从植物提取物的去除

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

从甘蓝型油菜中提取3页蛋白质和核糖配合物的韧皮部汁液

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing ...