This work was supported by a National Science Foundation Grant NSF-MCB1243645 (JZT), any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

注:可诱导的LAP标记的稳定细胞株为任何感兴趣的蛋白质的产生的概述在图1中被示出和LAP标记蛋白的表达，纯化和大规模蛋白质制剂的概要分析在图3中示出。 1.克隆兴趣的基因的开放阅读框（ORF）到LAP-标签矢量<ol><li>克隆兴趣的基因的ORF到穿梭载体。 <ol><li>使用聚合酶链反应（PCR），以扩增感兴趣的ORF用引物内的适当attB1和attB2位点对是N-端融合或C端融合。 见表1对PCR条件引物序列和表2中 。 </li><li>凝胶通过解决这些问题，在1％琼脂糖凝胶纯化PCR产物。切除扩增带，它是从凝胶中的正确的大小和使用DNA得自凝胶切片提取它埃尔提取试剂盒根据制造商的说明。 </li><li>孵育含有PCR产物与含有穿梭载体的attP位并允许PCR产物重组到载体中按照制造商的说明重组的纯化的attB位（参见主材料板表 ）。 注:空穿梭载体和LAP-标记载体包含的CCD B基因在CCD B抗性的细菌细胞中传播（见材料表 ）。然而CCDB基因重组出时的ORF插入这些载体中，从而用克隆的ORF传播矢量时使用标准的DH5α 大肠杆菌细胞。 </li><li>转化DH5α 大肠杆菌细胞用1μl的反应产物和板转化细胞到的Luria肉汤（LB）琼脂平板上以50毫克/毫升卡那霉素的13。 </li><li>选择卡那霉素抗性菌落。 </li><li>生长在LB我所选择的菌落直径为50毫克/毫升卡那霉素，使的DNA小量制备和使用表3中列出的测序引物通过DNA测序确认基因整合。 </li></ol></li><li>从传送穿梭载体兴趣的基因导入LAP-标签矢量。 <ol><li>孵育含有感兴趣ORF的序列经过证明的基因与LAP-标签载体（pGLAP1对于N末端融合）和介导的目的基因的从穿梭载体转移入LAP-标签矢量按照所述重组穿梭载体制造商的说明（参见材料）。 注:一系列可根据所需的启动子，表位标签被用于LAP / TAP矢量，和N-或C-末端标记可以在表4中找到。 </li><li>转化DH5α 大肠杆菌细胞用1μl的反应产物和板转化细胞上的LB琼脂板上，以100毫克/毫升氨苄青霉素13。 </li><li>选择氨苄青霉素抗性结肠新兴工业化经济体。 </li><li>生长在LB培养基将该选出的菌落用100毫克/毫升氨苄青霉素，使的DNA小量制备，并使用表5中所列的测序引物通过DNA测序确认基因整合。 </li></ol></li></ol> 2.生成诱导型稳定的细胞系，表达利益的LAP标记基因<ol><li>选择最适合于所关注的项目的细胞系。可替代地，创建从组成型表达的四面体和含有FRT位点，允许感兴趣的LAP标记基因被稳定地整合进基因组（参见材料）的任何现有的细胞系的宿主细胞系。 注意:此协议使用包含四面体和FRT位点，这是在-Tet的DMEM / F12培养基4 [用10％胎牛血清（FBS），该不含四环素（-Tet）制成]中生长的HEK293细胞系。 </li><li>确定潮霉素的最小浓度必须在1个宿主细胞系杀死2凌晨药品加成后，KS。浓度可宿主细胞系之间变化，以100微克/毫升和800微克/毫升之间最测距。 注:在37℃和5％的CO 2将在1-2周内死亡在-Tet的DMEM / F12培养基中生长，100微克/毫升潮霉素HEK293细胞。 </li><li>共转染表达所述翻转酶的重组酶使用转染试剂（介导的兴趣到细胞的基因组中的FRT位点的LAP标记基因的整合）与LAP标记载体导入HEK293细胞的载体按制造商的说明。使用的4比:重组酶载体的1至LAP矢量14。 注:最佳比例依赖于转染的宿主细胞系和方法，并可能需要一个滴定。 4:1的比例适用于大多数细胞系。包括一个模拟转板作为阴性对照。 </li><li>有一天，转染后，用新鲜的介质替换-Tet DMEM / F12培养基。 </li><li>两天后transfecti上分裂细胞25％汇合。允许细胞〜5小时附着，然后在2.2步一定的浓度添加含潮霉素-Tet DMEM / F12培养基。对HEK293细胞用100μg/ ml的潮霉素。 注:含有HEK293细胞系的FRT还包含与杀稻瘟抗性相关，因此在稳定细胞系的选择过程中使用5微克/毫升杀稻瘟选择用于四面体，并尽量减少渗漏表达的四面体。 </li><li>更换含潮霉素-Tet DMEM / F12媒体直到需要不同的细胞灶出现类似于对透明板不透明的斑。 </li><li>加入20微升胰蛋白酶对每一个细胞灶顶部和吸管上下2次用200μl枪头。位置细胞在24孔板并展开在含有潮霉素-Tet的DMEM / F12培养基的细胞不断生长。 </li><li>屏幕细胞诱导LAP-标记的蛋白质表达固定细胞或活细胞荧光显微镜和/或Western印迹的LAP-标签15中的GFP标记。 </li></ol> 3. LAP标记的蛋白复合物的分离纯化注:用于基于先前的经验的典型LAP标签蛋白纯化条件和卷下LAP标签蛋白纯化协议细节的建议。然而，应谨慎行事，以确保实验优化的利息蛋白复合物及蛋白表达水平，以提供最好的结果进行。 <ol><li>细胞生长和细胞收获。 <ol><li>扩大为蛋白质复合物的TAP隔离的验证LAP标记细胞系，由所有的HEK293细胞不断传代入-Tet的DMEM / F12介质更大的板和/或滚瓶在37℃和5％的CO 2。 </li><li>对于四环素/阿霉素诱导的细胞系，诱导10-15小时在0.2微克/毫升四环素/ DOX当细胞达到〜70％汇合的浓度harvestin前G细胞。 注:浓度和诱导时间应确定每个蛋白质，建议0.1-1微克/毫升四环素/ DOX的10-15小时的滴定。 </li><li>收获细胞通过搅拌或胰蛋白酶消化和沉淀的细胞以875 xg离心5-10分钟。 </li></ol></li><li>耦合抗GFP抗体蛋白A珠<ol><li>从0.5 ml细胞沉淀制备的裂解物中，红细胞压积（PCV）的使用抗体的40微克进行免疫沉淀。 注:将取决于大量的LAP-标记的蛋白质，以及其他因素的需要的抗体的量，并且将需要优化。推荐10-40微克的滴定。 </li><li>平衡160微升填充体积（PV）的蛋白A珠成的PBST（PBS + 0.1％吐温20）在1.5ml管中。用1ml的PBST洗涤3次。 注意:本文中所有的洗涤液通过在5000 xg离心离心珠子，持续10秒进行。 </li><li>珠悬浮在500微升PBST并加入80微克亲和力- 纯化的兔抗GFP抗体的160微升小珠每个管。混合，在室温下（RT）1小时。 </li><li>用1ml的PBST洗涤珠2次。然后，洗涤​​珠子2次，用1ml的0.2M硼酸钠，pH值9（20毫升0.2M的硼酸钠+15毫升的0.2M硼酸）的。最后一次洗涤后，加入900微升硼酸0.2M的钠，pH为9，使最终体积至1ml。 </li><li>加入100微升220毫dimethylpimelimidate（DMP）的为20毫米的最终浓度。轻轻旋转试管在室温30分钟。对于220毫米DMP，悬浮1个50毫克一瓶内容877微升0.2M的硼酸钠，pH值9，并立即加入到珠悬浮液。 </li><li>与DMP温育后，洗涤珠子1次用1毫升的0.2M乙醇胺，0.2M NaCl的pH 8.5的灭活残留交联剂。在1ml的相同缓冲液重悬珠子旋转在RT 1小时。沉淀珠粒并在500微升的0.2M乙醇胺，0.2M NaCl的pH 8.5的悬浮珠粒。珠是Severa的稳定升个月在4℃。 </li></ol></li><li>缓冲区的细胞裂解复杂的纯化制备方法<ol><li>通过使pH为7.4的溶液;制备LAPX缓冲器（300毫对于细胞裂解，200mM的大多数珠粒洗涤，和100mM洗涤珠粒之前洗脱的蛋白质，其中X为所需的盐的浓度[毫米氯化钾]的LAP缓冲器的）含有50mM 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）中，X毫米氯化钾，1mM的乙二醇四乙酸（EGTA），1mM的MgCl 2的，和10％甘油。 注:此缓冲区的组件用于在活细胞来近似环境。 HEPES用作在7.2-8.2的pH值愤怒的缓冲器。氯化钾是用于维持缓冲液的离子强度的盐。 EGTA是结合的钙离子，以减少相比镁钙的水平螯合剂。甘油和MgCl 2的用于提高蛋白质的稳定性。 </li><li>通过添加0.05％壬phenoxypol准备LAPX N缓冲yethoxylethanol到LAPX缓冲区。 注:壬phenoxypolyethoxylethanol是温和的洗涤剂增溶的蛋白质，但保留蛋白质 - 蛋白质相互作用，从而在提取过程中使用较高的浓度，然后将其在结合和洗涤步骤降低。 </li></ol></li><li>细胞裂解液的制备<ol><li>悬浮500微升PCV成2.5毫升LAP300的用0.5mM二硫苏糖醇（DTT）和蛋白酶抑制剂。加入90微升10％壬phenoxypolyethoxylethanol（0.3％最终），并通过倒置混合。 </li><li>在冰上放置10分钟。离心在21000 XG 10分钟。收藏本低速上清液（LSS）。就拿LSS的凝胶分析10微升的样品。 </li><li>在4℃LSS转移到TLA100.3管旋在100,000 XG 1小时。收藏本高速上清液（HSS），在管置于冰上。就拿HSS的凝胶分析10微升的样品。 注意:避免采取最上面的脂质层的D中的底部最细胞碎片层。脂质层应由之前收集所述HSS真空抽吸除去。 </li></ol></li><li>首先亲和捕获:绑定到抗GFP珠<ol><li>预洗脱抗体偶联的珠子（用160微升每0.5ml细胞沉淀（PCV）珠）由1毫升洗脱缓冲液[3.5米氯化镁 20毫米的Tris，pH值7.4的洗完3次摆脱解耦抗体和降低背景。快速完成。不要留在高盐珠子很长一段时间。 </li><li>用1ml的LAP200Ñ洗涤珠粒3次。混合HSS与抗体珠提取1小时，在4℃。离心在21000 XG 10分钟。取上清的10微升样品（ 即通过（FT）的流量）进行凝胶分析。 </li><li>用1ml的LAP200Ñ用0.5mM DTT和蛋白酶抑制剂洗珠子3次。用1ml的LAP200Ñ用0.5mM DTT和蛋白酶抑制剂的洗涤珠2次（每次5分钟）。快洗2吨输入法编辑器与用0.5mM DTT加入1ml LAP200 N和添加烟草蚀刻病毒（TEV）蛋白酶之前没有蛋白酶抑制剂。 </li></ol></li><li> TEV切割<ol><li>稀释为10μgTEV蛋白酶在1ml LAP200 N和在4℃旋转管过夜。 注意:此步骤可以为任何LAP标记蛋白进行优化，以在几个小时内，通过调整TEV蛋白酶的浓度，这可以帮助LAP标记蛋白质复合物的保存完成。 </li><li>粒料珠和转移上清液至新管。用160微升LAP200Ñ用0.5mM DTT和蛋白酶抑制剂（三重浓度）冲洗珠子两次，以除去任何残留的蛋白。取上清液进行凝胶分析的10微升样品。 </li></ol></li><li>二亲和捕获:绑定到的S蛋白琼脂糖<ol><li>洗80微升S蛋白琼脂糖浆液1管（40微升填充树脂）用1ml LAP200 N 3次。 注:•PROTEIN结合到S-标签将重组活性RNA酶和一个替代性的第二表位标签应考虑含RNA的蛋白质的复合物。 </li><li>添加TEV在4℃洗脱上清液S蛋白琼脂糖珠岩3小时。沉淀小珠和洗涤3次用1毫升LAP200Ñ用0.5mM DTT和蛋白酶抑制剂。用1毫升LAP100洗珠2次。 </li></ol></li><li>蛋白质洗脱<ol><li>通过加入50μl4×Laemmli样品缓冲液和热的在97℃下进行10分钟的琼脂糖洗脱蛋白质关S蛋白。 注:蛋白质，也可以从用洗脱缓冲液（3.5米的MgCl 2用20mM Tris，pH 7.4）中的珠洗脱。 </li></ol></li></ol> 4.确定通过质谱分析相互作用的蛋白质<ol><li>通过分析通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），银染凝胶的收集的样品测试纯化的质量（见<strong&gt;的材料表），并通过免疫印迹洗脱液并用抗GFP抗体探测，以确保LAP标记纯化工作16， 见图4。 </li><li>以确定化学计量和亚化学计量的共同净化物种，采取最终洗脱样品并通过SDS-PAGE分离它。染色用质谱兼容蛋白染色的凝胶。切除最突出的带，并从凝胶中它们之间的空间并对其进行处理用于通过质谱法分别16分析。 注:有分离最终纯化洗脱液，并为他们准备质谱5无数的方法。例如，LAP-纯化络合物可以由S蛋白珠通过使用高盐（3.5米的MgCl 2）和整个蛋白群集体可以通过质谱法17进行分析洗脱。可替换地，最终洗脱物可以1毫米×SDS-PAGE和一个1 mm表带能电子被分离xcised和分析。这将清除的任何珠粒或颗粒物质，将干扰分析的复杂混合物。 </li></ol>

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<li>Senese, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+unique+insertion+in+STARD9%27s+motor+domain+regulates+its+stability%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">A unique insertion in STARD9's motor domain regulates its stability.</a> Mol Biol Cell. 26, 440-452 (2015).</li>
<li>Gholkar, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+X-Linked-Intellectual-Disability-Associated+Ubiquitin+Ligase+Mid2+Interacts+with+Astrin+and+Regulates+Astrin+Levels+to+Promote+Cell+Division%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">The X-Linked-Intellectual-Disability-Associated Ubiquitin Ligase Mid2 Interacts with Astrin and Regulates Astrin Levels to Promote Cell Division.</a> Cell Rep. 14, 180-188 (2016).</li>
<li>Graumann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applicability+of+tandem+affinity+purification+MudPIT+to+pathway+proteomics+in+yeast%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast.</a> Mol Cell Proteomics. 3, 226-237 (2004).</li>
<li>Connolly, J. A., Kalnins, V. I., Cleveland, D. W., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunoflourescent+staining+of+cytoplasmic+and+spindle+microtubules+in+mouse+fibroblasts+with+antibody+to+tau+protein%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Immunoflourescent staining of cytoplasmic and spindle microtubules in mouse fibroblasts with antibody to tau protein.</a> Proc Natl Acad Sci U S A. 74, 2437-2440 (1977).</li>
<li>Gholkar, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tctex1d2+associates+with+short-rib+polydactyly+syndrome+proteins+and+is+required+for+ciliogenesis%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Tctex1d2 associates with short-rib polydactyly syndrome proteins and is required for ciliogenesis.</a> Cell Cycle. 14, 1116-1125 (2015).</li>
<li>Cheung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+Analysis+of+the+Mammalian+Katanin+Family+of+Microtubule-severing+Enzymes+Defines+Katanin+p80+subunit+B-like+1+%28KATNBL1%29+as+a+Regulator+of+Mammalian+Katanin+Microtubule-severing%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Proteomic Analysis of the Mammalian Katanin Family of Microtubule-severing Enzymes Defines Katanin p80 subunit B-like 1 (KATNBL1) as a Regulator of Mammalian Katanin Microtubule-severing.</a> Mol Cell Proteomics. 15, 1658-1669 (2016).</li>
<li>Torres, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+STARD9%2FKif16a+Kinesin+Associates+with+Mitotic+Microtubules+and+Regulates+Spindle+Pole+Assembly%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The STARD9/Kif16a Kinesin Associates with Mitotic Microtubules and Regulates Spindle Pole Assembly.</a> Cell. 147, 1309-1323 (2011).</li>
<li>Garcia-Gonzalo, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transition+zone+complex+regulates+mammalian+ciliogenesis+and+ciliary+membrane+composition%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition.</a> Nat Genet. 43, 776-784 (2011).</li>
<li>Chih, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+ciliopathy+complex+at+the+transition+zone+protects+the+cilia+as+a+privileged+membrane+domain%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain.</a> Nat Cell Biol. 14, 61-72 (2012).</li>
<li>Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S., Huang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+scalable+strategy+for+high-throughput+GFP+tagging+of+endogenous+human+proteins%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">A scalable strategy for high-throughput GFP tagging of endogenous human proteins.</a> Proc Natl Acad Sci U S A. 113, E3501-E3508 (2016).</li>
<li>Poser, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BAC+TransgeneOmics%3A+a+high-throughput+method+for+exploration+of+protein+function+in+mammals%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.</a> Nat Methods. 5, 409-415 (2008).</li>
<li>Firat-Karalar, E. N., Stearns, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+mammalian+centrosome+structure+using+BioID+proximity-dependent+biotinylation%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Probing mammalian centrosome structure using BioID proximity-dependent biotinylation.</a> Methods Cell Biol. 129, 153-170 (2015).</li>
<li>Roux, K. J., Kim, D. I., Burke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BioID%3A+a+screen+for+protein-protein+interactions%5BTitle%5D)+AND+%22Curr+Protoc+Protein+Sci%22%5BJournal%5D)">BioID: a screen for protein-protein interactions.</a> Curr Protoc Protein Sci. 74, (2013).</li>
<li>Gupta, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Dynamic+Protein+Interaction+Landscape+of+the+Human+Centrosome-Cilium+Interface%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface.</a> Cell. 163, 1484-1499 (2015).</li>
</ol>

The authors have nothing to disclose.

概述的协议描述了感兴趣的基因的克隆到LAP标记向量，生成诱导LAP标记的稳定细胞株，以及LAP标记蛋白质复合物的蛋白质组分析的纯化。对于其他LAP / TAP标记的方法，该协议已被简化为与高通量方法的细胞通路中映射蛋白定位和蛋白质 - 蛋白质相互作用兼容。这种方法已被广泛应用到为细胞周期进程，有丝分裂纺锤体组装，纺锤体极稳态，和ciliogenesis临界仅举几蛋白质的功能特性，并且辅助的这些蛋白质的误调节如何能导致人类疾病15的理解， 16,19,20。例如，我们的小组最近利用这个系统来定义纺锤体组装15,21的中STARD9丝分裂驱动蛋白的功能和调节（候选癌症的目标），定义在Tctex1d2动力蛋白轻链之间的新型分子联系 和短肋多指畸形综合征（SRPS）19，并确定了新的分子联系，以了解MID2泛素连接酶突变如何导致X-连锁智力残疾16。其他实验室还成功地应用这个方法，其中包括一个确定Tctn1，小鼠Hedgehog信号的调节，是一个ciliopathy相关蛋白复合物，的一部分以依赖组织方式22,23稳压睫状膜组成和ciliogenesis。因此，此协议可以广泛地应用到任何细胞途径的解剖。 在本协议中的一个关键步骤是那些潮霉素抗性LAP-标记的稳定细胞系的选择。特别应注意，以确保在控制板上的所有细胞是在实验板上放大选择灶之前死亡。潮霉素也可以ADDE的LAP标记的稳定细胞株常规细胞培养期间d，来进一步确保所有细胞维持感兴趣的LAP-标记基因在FRT位点。我们告诫，并非所有的LAP标记的蛋白质将是正常，并且它具有在位置测定可用于测试蛋白质的功能是重要的。用于测试蛋白质的功能测定法的实例包括siRNA的诱导表型和体外活性测定法救援。为了解决与另外一个大LAP-标签的任何潜在的问题，我们先前已经产生与该系统包含小的标记，如FLAG，这是不太可能抑制所感兴趣的蛋白的功能和定位兼容的TAP标记的载体4。此外，用于产生C-末端LAP标记蛋白或C末端的TAP标记的蛋白质与本系统，可以在的情况下使用，其中一个LAP / TAP标记不在在N耐受兼容存在LAP标记矢量蛋白质的-terminus。此外，日纯化缓冲器（LAPX N）的电子盐和洗涤剂浓度可以被修饰以提高或如果观察到无或过多的相互作用降低纯化严格性。类似地，串联亲和纯化过程比单一纯化步骤和弱相互作用物更严格的可能丢失，从而可以在很少或没有相互作用物被识别用于一个单一的纯化方案。 要注意的是其他的GFP的表位标记的方法存在允许大规模的GFP蛋白标记的蛋白质的定位和纯化研究24,25是重要的。这些包括上述BAC TransgenOmics方法，利用细菌人工染色体从其天然环境表达感兴趣GFP标记基因包含所有调节元件，该模拟内源基因表达24。最近，CAS9 /单引导RNA（因组）核糖核蛋白复合物（的RNP）已经被用于结束ogenously标记的有分流-GFP系统，使从它们的内源基因位点25的GFP标记基因的表达感兴趣的基因。虽然这两种方法使标记的蛋白质的内源性的条件下，表达相比，这里描述的LAP标记协议，它们不允许针对感兴趣的标记基因的诱导和可调表达。此外，他们还没有被应用到串联表位标记的TAP。同样重要的是要注意，其他标签系统也可以被修改以成为与在此描述用于产生诱导的表位标记的稳定细胞株的系统兼容。例如，接近度依赖生物素标识（BioID）已经获得相当多的关注，因为它能够相互作用蛋白26之间限定的空间和时间关系的能力。这种技术利用蛋白质融合到大肠杆菌生物素连接酶生物素化酶BirA的混杂菌株，其中生物素化S中的酶的〜10nm的范围内的任何蛋白质。生物素化的蛋白，然后亲和使用生物素亲和捕获纯化和通过质谱法的组合物进行分析。生物素化酶BirA将生物素化的任何蛋白质在接近，甚至短暂，这使得它特别适合于复杂的27内检测弱相互作用伙伴。此外，纯化方案并不需要内源性蛋白质 - 蛋白质相互作用保持不变，并可以在变性条件下进行，从而降低假阳性率。在我们目前的协议，通过生物素化酶BirA标记矢量的pGLAP1向量的置换可以从确定基于亲和力基于接近检测它们的蛋白质 - 蛋白质相互作用变换这个系统。因为是许多酶 - 底物的相互作用之间并用于映射时空蛋白质INTE的情况下这样的系统将是，用于检测瞬时蛋白质相互作用是非常有利的作为中心体和纤毛26,28已进行了限定的结构内ractions。

To investigate the cellular function of an uncharacterized protein it is important to determine its in vivo spatiotemporal subcellular localization and its interacting protein partners. Traditionally, single and tandem epitope tags fused to the N or C-terminus of a protein of interest have been used to facilitate protein localization and protein interaction studies. For example, the tandem affinity purification (TAP) technology has enabled the isolation of native protein complexes, even those that are in low abundance, in both yeast and mammalian cell lines1,2. The localization and affinity purification (LAP) technology, is a more recent development that modifies the TAP procedure to include a localization component through the introduction of the green fluorescent protein (GFP) as one of the epitope tags3. This approach has given researchers a deeper understanding of a protein's subcellular localization in living cells while also retaining the ability to perform TAP complex purifications to map protein-protein interaction networks. 
However, there are many issues associated with the use of TAP/LAP technologies that has hampered their widespread use in mammalian cells. For example, the length of time that is necessary to generate a stable cell line expressing a TAP/LAP tagged protein of interest; which typically relies on cloning the gene of interest into a viral vector and selecting single cell stable integrants with the desired expression level. Additionally, many cellular pathways are sensitive to constitutive protein overexpression (even at low levels) and can arrest cells or trigger cell death over time making the generation of a TAP/LAP stable cell line impossible. These and other constraints have impeded LAP/TAP methodologies from becoming high-throughput systems for protein localization and protein complex elucidation. Therefore, there has been considerable interest in the development of an inducible high-throughput LAP-tagging system for mammalian cells that takes advantage of current innovations in cloning and cell line technologies. 
Here we present a protocol for generating stable cell lines with Doxycycline/Tetracycline (Dox/Tet) inducible LAP-tagged proteins of interest that applies advances in both cloning and mammalian cell line technologies. This approach streamlines the acquisition of data with regards to LAP-tagged protein subcellular localization, protein complex purification and identification of interacting proteins4. Although affinity proteomics utilizes a wide range of techniques for protein complex elucidation5, our approach is beneficial for expediting the identification of these complexes and their native interaction networks and is amenable to high-throughput protein tagging that is necessary to investigate complex biological pathways that contain a multitude of protein constituents. Key to this approach are advancements in cloning strategies that enable high fidelity and expedited cloning of target genes into an array of vectors for gene expression in vitro, in various organisms like bacteria and baculovirus, and in mammalian cells6,7. Additionally, the ORFeome collaboration has cloned thousands of sequence validated open reading frames in vectors that incorporate these advances in cloning, which are available to the scientific community8-11. In our system, the pGLAP1 LAP-tagging vector enables the simultaneous cloning of a large number of clones, which facilitates high-throughput LAP-tagging. This expedited cloning procedure is coupled to a streamlined approach for generating cell lines with LAP-tagged genes of interest inserted at a single pre-determined genomic locus. This makes use of cell lines that contain a single flippase recognition target (FRT) site within their genome, which is the site of integration for LAP-tagged genes. These cell lines also express the tetracycline repressor (TetR) that binds to Tet operators (TetO2) upstream of the LAP-tagged genes and silences their expression in the absence of Dox/Tet. This allows for Dox/Tet inducible expression of the LAP-tagged protein at any given time. Having the capability of inducible LAP-tagged protein expression is critical, since many cellular pathways are sensitive to the levels of critical proteins governing the pathway and can arrest cell growth or trigger cell death when these proteins are constitutively overexpressed, even at low levels, making the generation of non-inducible LAP-tagged stable cell lines impossible12.

<table><tbody><tr><td>Flp-In T-REx Core Kit</td><td>Invitrogen</td><td>K6500-01</td><td>Kit for generating cell lines that contain an FRT site and TrtR expression</td></tr><tr><td>PETG, 5x</td><td>Nunc, Inc.&amp;nbsp;</td><td>73520-734</td><td>Roller bottle for growing cells</td></tr><tr><td>PETG, 2.5x</td><td>Nunc, Inc.&amp;nbsp;</td><td>73520-420</td><td>Roller bottle for growing cells</td></tr><tr><td>Cell stackers</td><td>Corning CellSTACK</td><td>3271</td><td>Cell stacker for growing cells</td></tr><tr><td>500 ml conical centrifuge tubes</td><td>Corning&amp;nbsp;</td><td>431123</td><td>Tubes for harvesting cells</td></tr><tr><td>Anti-GFP antibody</td><td>Invitrogen</td><td>A11122</td><td>Rabbit anti GFP antibody</td></tr><tr><td>Affiprep Protein A beads</td><td>Biorad</td><td>156-0006</td><td>Used as a matrix for conjugating anti-GFP antibodies</td></tr><tr><td>Dimethylpimelimidate (DMP)</td><td>ThermoFisher Scientific</td><td>21667</td><td>Used for conjugating anti-GFP antibodies to Protein A beads</td></tr><tr><td>TLA100.3 tubes</td><td>Beckman</td><td>349622</td><td>Tubes for centrifuging protein lysates during the clearing step</td></tr><tr><td>TEV protease</td><td>Invitrogen</td><td>12575-015</td><td>Used for cleaving the GFP tag off of N-terminal LAP-tagged proteins</td></tr><tr><td>Precession Protease</td><td>GE Healthcare</td><td>27-0843-01</td><td>Used for cleaving the GFP tag off of C-terminal LAP-tagged proteins</td></tr><tr><td>S-protein agarose</td><td>Novagen</td><td>69704</td><td>Used as a second affinity matrix during the purification of LAP-tagged protein complexes</td></tr><tr><td>QIAquick DNA gel extraction kit</td><td>Qiagen</td><td>28704/28706</td><td>For use in purifying PCR products from an agarose gel</td></tr><tr><td>BP clonase II</td><td>Invitrogen</td><td>11789020</td><td>Used for cloning ORF PCR products into the pDONR221 shuttle vector</td></tr><tr><td>LR clonase II</td><td>Invitrogen</td><td>11791020</td><td>Used for cloning the ORF of the gene of interest into the pGLAP1 LAP-tagging vector</td></tr><tr><td>&lt;em&gt;ccdB&lt;/em&gt; Survival 2 T1&lt;sup&gt;R&lt;/sup&gt; &lt;em&gt;E. coli&lt;/em&gt;</td><td>Invitrogen</td><td>A10460</td><td>Used for propgating shuttle vectors and pGLAP empty vectors</td></tr><tr><td>Fugene 6</td><td>Promega</td><td>E2691</td><td>Transfection reagent for transfecting vectors into human cells</td></tr><tr><td>Tetracycline</td><td>Invitrogen</td><td>Q100-19</td><td>Drug for inducing Dox/Tet inducible protein expression&amp;nbsp;</td></tr><tr><td>Doxycycline</td><td>Clontech</td><td>631311</td><td>Drug for inducing Dox/Tet inducible protein expression&amp;nbsp;</td></tr><tr><td>Hygromycin B</td><td>Invitrogen</td><td>10687010</td><td>Drug for selecting stable LAP-tagged integrants</td></tr><tr><td>Kanamycin</td><td>Corning</td><td>61-176-RG</td><td>Drug for selecting Kanamycin resistant bacterial colonies</td></tr><tr><td>Ampicillin</td><td>Fisher</td><td>BP1760-5</td><td>Drug for selecting Ampicillin resistant bacterial colonies</td></tr><tr><td>4-20% Tris Glycine SDS-PAGE gels</td><td>Biorad</td><td>4561094</td><td>Used for separating protein samples and final LAP-tag purification eluates</td></tr><tr><td>Silver Stain Plus Kit</td><td>Biorad</td><td>1610449</td><td>Used for silver staining the eluates of LAP-tagged pufications and samples collected throughout the purification process&amp;nbsp;</td></tr><tr><td>Coomassie Blue stain</td><td>Invitrogen</td><td>LC6060</td><td>Used for staining SDS-PAGE gels to visulize LAP-tagged purifications and cutting out protein bands, mass spectrometry compatible</td></tr><tr><td>Shuttle vector pDONR221</td><td>Invitrogen</td><td>12536017</td><td>Shuttle vector for cloning the ORFs of genes of interest</td></tr><tr><td>Flippase expressing vector pOG44</td><td>Invitrogen</td><td>V600520</td><td>Vector that expresses the Flippase recombinase for integrating LAP-tagged genes into the genome of FRT site containing cell lines</td></tr><tr><td>Platinum Taq DNA Polymerase</td><td>ThermoFisher Scientific</td><td>10966018</td><td>Used for PCR amplification of the ORFs of genes of interest</td></tr><tr><td>4x Laemmli sample buffer</td><td>Biorad</td><td>1610747</td><td>Sample buffer for eluting purified LAP-tagged protein complexes from the bead matrix</td></tr><tr><td>Luria broth (LB) media</td><td>Fisher</td><td>BP9723-2</td><td>Used for growing DH5&amp;alpha; bacteria</td></tr><tr><td>DNA miniprep kit</td><td>Promega</td><td>A1222</td><td>Used for making DNA plasmid minipreps</td></tr><tr><td>DMEM/F12 media</td><td>Hyclone</td><td>SH30023.01</td><td>For growing Hek293 human cells</td></tr><tr><td>FBS lacking Tet</td><td>Altanta Biologicals</td><td>S10350</td><td>Used for making -Tet DMEM/F12 media for generating and growing inducible LAP-tagged stable cell lines</td></tr><tr><td>Trypsin</td><td>Hyclone</td><td>SH30042.01</td><td>For lifting Hek293 cell foci from plates</td></tr><tr><td>Protease inhibitor tablets</td><td>Roche</td><td>11836170001</td><td>Used for making protocol buffers, EDTA-free</td></tr><tr><td>10% nonyl phenoxypolyethoxylethanol&amp;nbsp;</td><td>Roche</td><td>11332473001</td><td>Used for making protocol buffers</td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CM</td><td>Used for making protocol buffers</td></tr><tr><td>Tween-20</td><td>Fisher</td><td>BP337-500</td><td>Used for making protocol buffers</td></tr><tr><td>Sodium Borate</td><td>Fisher</td><td>S249-500</td><td>Used for making protocol buffers</td></tr><tr><td>Boric Acid</td><td>Fisher</td><td>A78-500</td><td>Used for making protocol buffers</td></tr><tr><td>Ethanolamine</td><td>Calbiochem</td><td>34115</td><td>Used for making protocol buffers</td></tr><tr><td>NaCl</td><td>Fisher</td><td>P217-3</td><td>Used for making protocol buffers</td></tr><tr><td>KCl</td><td>Fisher</td><td>BP358-10</td><td>Used for making protocol buffers</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher</td><td>BP172-25</td><td>Used for making protocol buffers</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>M33-500</td><td>Used for making protocol buffers</td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP152-5</td><td>Used for making protocol buffers</td></tr></tbody></table>

inducible lap-tagged stable cell lines for investigating protein function, spatiotemporal localization and protein interaction networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

为了突出本系统的效用，牛头微管结合蛋白的开放读框（ORF）通过用含有attB1和attB2位点的引物扩增头的ORF（ 表1）和温育PCR产物与克隆入穿梭载体穿梭载体和介导的PCR产物插入到穿梭载体中的重组酶。反应产物被用来从卡那霉素抗性菌落变换DH5α菌13和质粒DNA进行测序，以确保头插入。然后，序列进行验证穿梭头载体使用时，通过与pGLAP1矢量孵育穿梭头载体的头的ORF转移到pGLAP1载体，其稠合头在帧与LAP（EGFP-TEV-S蛋白）标记和介导的ORF从穿梭载体来pGLAP1转印重组酶。该反应产物用于转化DH5α细菌从氨苄青霉素抗性菌落13和质粒DNA进行测序，以确保LAP-头融合在框架上。序列验证pGLAP1-LAP-头，然后共转染用表达所述翻转酶的重组酶入HEK293细胞含有单一的翻转酶识别目标的基因组中（FRT）位点，其是集成为LAP标记基因的位点的载体14。该细胞系还表示，结合春节运营商LAP-标记基因的上游和沉默在没有四环素/ DOX其表达四面体。稳定整合体选择具有-Tet的DMEM / F12培养基用100微克/毫升潮霉素5天。个别潮霉素耐药细胞灶加顶部20微升胰蛋白酶和上下吹打2次收获。细胞置于24孔板并在-Tet的DMEM / F12培养基持续增长扩大。 要验证潮霉素耐药细胞小号是能够表达LAP-头的，HEK293 LAP-头细胞用0.1微克/毫升阿霉素诱导的15小时和蛋白质提取物从非诱导和阿霉素诱导的细胞制备。这些提取物用SDS-PAGE分离，转移到PVDF膜，并进行免疫印迹对GFP和微管蛋白作为上样对照。如在图4A中 ，LAP-头看到（可视化与抗GFP抗体）仅在霉素的存在下表达。以验证LAP-头被正确地定位于有丝分裂过程中的有丝分裂的微管纺锤体，如先前已示出为内源性头18，HEK293 LAP-头细胞用0.1微克/毫升阿霉素诱导的15小时和固定细胞，用4％多聚甲醛和共染色的DNA（赫斯特33342）和微管（抗微管蛋白抗体）。一致，LAP-头被有丝分裂的中期（ 图4B）中定位于有丝分裂纺锤体。要验证LAP-头及其相互作用的蛋白质可能与此SYS提纯统，HEK293 LAP-头细胞滚瓶至〜70％汇合生长，用0.1微克/毫升阿霉素诱导的15小时，通过搅拌收获，用LAP300缓冲液裂解，并使用上述协议纯化LAP-头。从LAP-头纯化洗脱液用SDS-PAGE分离和将凝胶银染色。 图4C显示了LAP-头净化，标有星号是LAP-头和其他几个乐队指示头相互作用的蛋白质可以看出。 <img alt="图1" src="/files/ftp_upload/54870/54870fig1.jpg" /> 图1:LAP- 标记诱导稳定细胞系的利息的任何蛋白的生成概述。感兴趣的基因的开放阅读框（ORF）的扩增与attB1和attB2位点侧翼的5&#39;和3&#39;末端序列，（引物序列在给定的 表1），并克隆到<html>穿梭载体。与感兴趣的基因序列已验证穿梭载体然后用于感兴趣的基因转移到pGLAP1载体。验证pGLAP1载体用感兴趣的基因序列是然后共转染用含有翻转重组到一个包含单一翻转酶识别目标的基因组中（FRT）位点，其是集成为LAP的部位所需要的细胞系中的矢量-tagged基因。这些细胞系也表达了四环素阻遏（四面体）结合春节运营商（TETO 2）LAP-标记基因的上游和沉默在没有四环素/阿霉素的表达。然后感兴趣的LAP标记基因被重组到FRT位点和稳定的整合与潮霉素选择。 <a href="http://ecsource.jove.com/files/ftp_upload/54870/54870fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> 2"SRC ="/文件/ ftp_upload / 54870 / 54870fig2.jpg"/&gt; 图2: 对申请LAP标记的稳定细胞系概述。 LAP标记诱导的稳定细胞株被诱导表达的由阿霉素加成感兴趣LAP标签蛋白，并且可以在细胞周期的不同阶段进行同步，或者可以与化学品或配体刺激以激活任何所需的信号传导途径。感兴趣的LAP标签蛋白的亚细胞定位可以通过活细胞或固定细胞成像进行分析。 LAP标签的蛋白质也可以是串联亲和纯化，和它们的相互作用的蛋白质可以通过液相色谱串联质谱法（LC-MS / MS）来鉴定。最后，Cytoscape中可用于产生诱饵蛋白的蛋白 - 蛋白相互作用网络。 DOX表示强力霉素，IP表示免疫沉淀，EGFP表示增强型绿色荧光蛋白，Tev来表示TEV蛋白酶切割位点，S表示S标签。http://ecsource.jove.com/files/ftp_upload/54870/54870fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54870/54870fig3.jpg" /> 图3:LAP- 标记蛋白的表达，纯化和制备质谱的概述。该协议具有9个步骤:1）LAP标记蛋白表达的生长和诱导，2）细胞收获和裂解，3）制备的裂解物的，4）以抗GFP珠裂解物的结合，5）TEV蛋白酶裂解将GFP标签，6）的裂解物的至S蛋白珠结合，7）诱饵蛋白和相互作用蛋白，以及8-9）对于基于质谱的蛋白质组分析的样品的制备的洗脱。 <a href="http://ecsource.jove.com/files/ftp_upload/54870/54870fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p&gt; <img alt="图4" src="/files/ftp_upload/54870/54870fig4.jpg" /> 图4:LAP- 头表达的验证。 （A）中Western印迹（WB）从用抗GFP和抗微管蛋白抗体探测以检测LAP标记Tau蛋白和微管蛋白上样对照，分别未诱导和阿霉素诱导的LAP-头HEK293细胞蛋白质样品的分析。请注意，当细胞与阿霉素诱导的LAP-头只表述。表达LAP-头（B）的有丝分裂细胞固定并共染色用抗微管蛋白抗体和LAP-tau蛋白的亚细胞定位进行DNA（赫斯特33342）和微管蛋白（浴缸）通过荧光显微术进行分析。注意，LAP-头有丝分裂过程中定位于有丝分裂纺锤体和纺锤体极。 （C）银染色LAP-头净化凝胶。 MW表示分子量，CL表示清除裂解液和E印度语阿泰最终洗脱液。样品在4-20％SDS-PAGE上运行，并将凝胶银染以显现纯化的蛋白质。注意，对应于LAP-头的频带被标有星号和对应于共同纯化的蛋白质可以看出其他几个频带。 <a href="http://ecsource.jove.com/files/ftp_upload/54870/54870fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> N-末端融合 </td><td></td></tr><tr><td>前锋</td><td> 5&#39;- GGGGACAAGT TTGTACAAAAAAGCAGGCT TCATG - （&gt; 18gsn）-3&#39; </td></tr><tr><td>相反</td><td> 5&#39;- GGGGACCACTTTGTACAAGAAAGCTGGGTŧTTATCA - （&gt; 18gsn）-3&#39; </td></tr><tr><td> C-末端融合 </td><td></td></tr><tr><td>前锋</td><td> 5&#39;- GGGGACAAGTTTGTACAAAAAAGCAGGCT TCACC - （&gt; 18gsn）-3&#39; </td></tr><tr><td>相反</td><td> 5&#39;- GGGGACCACTTTGTACAAGAAAGCTGGGT摹- （&gt; 18gsn）-3&#39; </td></tr></tbody></table> 表1:正向和反向放大的ORF或利息插入穿梭载体引物。的attB位点以粗体字母突出显示，GSN表示超过18的基因特异性核苷酸加入到引物。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"> 步 </td><td> 温度 </td><td> 时间 </td></tr><tr><td colspan="2">初始变性</td><td> 94℃ </td><td> 2分钟</td></tr><tr><td rowspan="3"> PCR扩增循环（35） </td><td>变性</td><td> 94℃ </td><td> 30秒</td></tr><tr><td>退火</td><td> 55℃（取决于引物的Tm） </td><td> 30秒</td></tr><tr><td>延伸</td><td> 72℃ </td><td> 1分钟/ kb </td></tr><tr><td colspan="2">保持</td><td> 4℃ </td><td>无限期</td></tr></tbody></table> 表2:PCR条件利息的ORF的扩增。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 向量 </td><td> 正向测序引物 </td><td> 反向测序引物 </td></tr><tr><td>穿梭载体</td><td> 5&#39;-TGTAAAACGACGGCCAGT-3&#39; </td><td> 5&#39;-CAGGAAACAGCTATGAC-3&#39; </td></tr></tbody></table> 表3:正向和反向测序引物的穿梭载体。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ID </td><td> 结构体 </td><td> 父母 </td><td> 促进者 </td><td> 北RES </td><td> 马姆RES </td><td> 春节章？ </td></tr><tr><td> pGLAP1 </td><td> N-末端EGFP-TEV-S肽</td><td>的pcDNA5 / FRT / TO </td><td> CMV </td><td>安培</td><td> HYG </td><td>是</td></tr><tr><td> pGLAP2 </td><td> N-末端旗TEV-S肽</td><td>的pcDNA5 / FRT / TO </td><td> CMV </td><td>安培</td><td> HYG </td><td>是</td></tr><tr><td> pGLAP3 </td><td> N-末端EGFP-TEV-S肽; C-V5长期</td><td> pEF5 / FRT-V5 </td><td> EF1A </td><td>安培</td><td> HYG </td><td>没有</td></tr><tr><td> pGLAP4 </td><td> N-末端旗TEV-S肽; ; C-V5长期</td><td> pEF5 / FRT-V5 </td><td> EF1A </td><td&gt;放大器</td><td> HYG </td><td>没有</td></tr><tr><td> pGLAP5 </td><td> C-相S肽PreProt X2-EGFP </td><td> pEF5 / FRT-V5 </td><td> EF1A </td><td>安培</td><td> HYG </td><td>没有</td></tr></tbody></table> 表4:具有可变促销员，表位标记，对于N霉素诱导表达能力或C端蛋白标记可用LAP / TAP向量列表。载体是市售的。北RES表明细菌耐药标记，MAM RES表明哺乳动物细胞抗性标记，春节章？指示表达式是否为春节/ DOX调控。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 向量 </td><td> 正向测序引物 </td><td> 反向测序引物 </td></tr><tr><td> pGLAP1 </td><td> 5&#39;-ATCACTCTCGGCATGGACGAGCTGTACAAG-3&#39; </td><td> 5&#39;-TGGCTGGCAACTAGAAGGCACAGTCGAGGC-3&#39; </td></tr><tr><td> pGLAP2 </td><td> 5&#39;-CGAACGCCAGCACATGGACAGGG-3&#39; </td><td> 5&#39;-TGGCTGGCAACTAGAAGGCACAGTCGAGGC-3&#39; </td></tr><tr><td> pGLAP3 </td><td> 5&#39;-AGAAACCGCTGCTGCTAA-3&#39; </td><td> 5&#39;-TAGAAGGCACAGTCGAGG-3&#39; </td></tr><tr><td> pGLAP4 </td><td> 5&#39;-AGACCCAAGCTGGCTAGGTAAGC-3&#39; </td><td> 5&#39;-TAGAAGGCACAGTCGAGG-3&#39; </td></tr><tr><td> pGLAP5 </td><td> 5&#39;-CGTAATACGACTCACTATAG-3&#39; </td><td> 5&#39;-TCCAGGGTCAAGGAAGGCACGG-3&#39; </td></tr></tbody></table> 表5:正向和反向测序引物为载体pGLAP。

Watch this Scientific Journal Video about Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks at JoVE.com

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

诱导LAP标记的稳定细胞系用于研究蛋白质功能，时空定位与蛋白质相互作用网络

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

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Research

JoVE Journal

Biology

9.8K 次观看.  University of California, Los Angeles. We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks. 

视频: Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

科研

生物化学

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

遗传学

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

通过旗-生物素串联纯化通过交叉链接和免疫沉淀对蛋白质-RNA相互作用进行转录组-全过程分析

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

发育生物学

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

免疫与感染

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

生物学

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

基于 TurboID 的接近标签，用于蛋白质-蛋白质相互作用网络的植物中识别

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.