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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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoflourescent+staining+of+cytoplasmic+and+spindle+microtubules+in+mouse+fibroblasts+with+antibody+to+tau+protein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tctex1d2+associates+with+short-rib+polydactyly+syndrome+proteins+and+is+required+for+ciliogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STARD9/Kif16a+Kinesin+Associates+with+Mitotic+Microtubules+and+Regulates+Spindle+Pole+Assembly.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transition+zone+complex+regulates+mammalian+ciliogenesis+and+ciliary+membrane+composition.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ciliopathy+complex+at+the+transition+zone+protects+the+cilia+as+a+privileged+membrane+domain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scalable+strategy+for+high-throughput+GFP+tagging+of+endogenous+human+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+TransgeneOmics:+a+high-throughput+method+for+exploration+of+protein+function+in+mammals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+mammalian+centrosome+structure+using+BioID+proximity-dependent+biotinylation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioID:+a+screen+for+protein-protein+interactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Dynamic+Protein+Interaction+Landscape+of+the+Human+Centrosome-Cilium+Interface.">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 border="0" cellpadding="0" cellspacing="0" width="640">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flp-In T-REx Core Kit</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">K6500-01</td>
			<td style="width:167px;">Kit for generating cell lines that contain an FRT site and TrtR expression</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PETG, 5X</td>
			<td style="width:139px;">Nunc, Inc.&#160;</td>
			<td style="width:119px;">73520-734</td>
			<td>Roller bottle for growing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PETG, 2.5X</td>
			<td style="width:139px;">Nunc, Inc.&#160;</td>
			<td style="width:119px;">73520-420</td>
			<td>Roller bottle for growing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell stackers</td>
			<td style="width:139px;">Corning CellSTACK</td>
			<td style="width:119px;">3271</td>
			<td>Cell stacker for growing cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">500 mL conical centrifuge tubes</td>
			<td style="width:139px;">Corning&#160;</td>
			<td style="width:119px;">431123</td>
			<td>Tubes for harvesting cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-GFP antibody</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A11122</td>
			<td>Rabbit anti GFP antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Affiprep Protein A beads</td>
			<td style="width:139px;">Biorad</td>
			<td style="width:119px;">156-0006</td>
			<td>Used as a matrix for conjugating anti-GFP antibodies</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dimethylpimelimidate (DMP)</td>
			<td style="width:139px;">ThermoFisher Scientific</td>
			<td>21667</td>
			<td>Used for conjugating anti-GFP antibodies to Protein A beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TLA100.3 tubes</td>
			<td style="width:139px;">Beckman</td>
			<td style="width:119px;">349622</td>
			<td>Tubes for centrifuging protein lysates during the clearing step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEV protease</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">12575-015</td>
			<td>Used for cleaving the GFP tag off of N-terminal LAP-tagged proteins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precession Protease</td>
			<td style="width:139px;">GE Healthcare</td>
			<td style="width:119px;">27-0843-01</td>
			<td>Used for cleaving the GFP tag off of C-terminal LAP-tagged proteins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">S-protein agarose</td>
			<td style="width:139px;">Novagen</td>
			<td style="width:119px;">69704</td>
			<td>Used as a second affinity matrix during the purification of LAP-tagged protein complexes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAquick DNA gel extraction kit</td>
			<td style="width:139px;">Qiagen</td>
			<td>28704/28706</td>
			<td>For use in purifying PCR products from an agarose gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BP clonase II</td>
			<td style="width:139px;">Invitrogen</td>
			<td>11789020</td>
			<td>Used for cloning ORF PCR products into the pDONR221 shuttle vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LR clonase II</td>
			<td style="width:139px;">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 height="23">
			<td height="23" style="height:23px;">ccdB Survival 2 T1R E. coli</td>
			<td style="width:139px;">Invitrogen</td>
			<td>A10460</td>
			<td>Used for propgating shuttle vectors and pGLAP empty vectors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fugene 6</td>
			<td style="width:139px;">Promega</td>
			<td>E2691</td>
			<td>Transfection reagent for transfecting vectors into human cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetracycline</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">Q100-19</td>
			<td>Drug for inducing Dox/Tet inducible protein expression&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Doxycycline</td>
			<td style="width:139px;">Clontech</td>
			<td style="width:119px;">631311</td>
			<td>Drug for inducing Dox/Tet inducible protein expression&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hygromycin B</td>
			<td style="width:139px;">Invitrogen</td>
			<td>10687010</td>
			<td>Drug for selecting stable LAP-tagged integrants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
			<td style="width:139px;">Corning</td>
			<td>61-176-RG</td>
			<td>Drug for selecting Kanamycin resistant bacterial colonies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:139px;">Fisher</td>
			<td>BP1760-5</td>
			<td>Drug for selecting Ampicillin resistant bacterial colonies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-20% Tris Glycine SDS-PAGE gels</td>
			<td style="width:139px;">Biorad</td>
			<td>4561094</td>
			<td>Used for separating protein samples and final LAP-tag purification eluates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silver Stain Plus Kit</td>
			<td style="width:139px;">Biorad</td>
			<td>1610449</td>
			<td>Used for silver staining the eluates of LAP-tagged pufications and samples collected throughout the purification process&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coomassie Blue stain</td>
			<td style="width:139px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">Shuttle vector pDONR221</td>
			<td style="width:139px;">Invitrogen</td>
			<td>12536017</td>
			<td>Shuttle vector for cloning the ORFs of genes of interest</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flippase expressing vector pOG44</td>
			<td style="width:139px;">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 height="40">
			<td height="40" style="height:40px;width:216px;">Platinum Taq DNA Polymerase</td>
			<td style="width:139px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10966018</td>
			<td>Used for PCR amplification of the ORFs of genes of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4X Laemmli sample buffer</td>
			<td style="width:139px;">Biorad</td>
			<td>1610747</td>
			<td>Sample buffer for eluting purified LAP-tagged protein complexes from the bead matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria broth (LB) media</td>
			<td style="width:139px;">Fisher</td>
			<td>BP9723-2</td>
			<td>Used for growing DH5&#945; bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA miniprep kit</td>
			<td style="width:139px;">Promega</td>
			<td>A1222</td>
			<td>Used for making DNA plasmid minipreps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 media</td>
			<td style="width:139px;">Hyclone</td>
			<td>SH30023.01</td>
			<td>For growing Hek293 human cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS lacking Tet</td>
			<td style="width:139px;">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 height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td style="width:139px;">Hyclone</td>
			<td>SH30042.01</td>
			<td>For lifting Hek293 cell foci from plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor tablets</td>
			<td style="width:139px;">Roche</td>
			<td style="width:119px;">11836170001</td>
			<td>Used for making protocol buffers, EDTA-free</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10% nonyl phenoxypolyethoxylethanol&#160;</td>
			<td style="width:139px;">Roche</td>
			<td style="width:119px;">11332473001</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:139px;">Corning</td>
			<td>21-040-CM</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td style="width:139px;">Fisher</td>
			<td>BP337-500</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Borate</td>
			<td style="width:139px;">Fisher</td>
			<td>S249-500</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td style="width:139px;">Fisher</td>
			<td>A78-500</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanolamine</td>
			<td style="width:139px;">Calbiochem</td>
			<td>34115</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:139px;">Fisher</td>
			<td>P217-3</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:139px;">Fisher</td>
			<td>BP358-10</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td style="width:139px;">Fisher</td>
			<td>BP172-25</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td style="width:139px;">Fisher</td>
			<td>M33-500</td>
			<td>Used for making protocol buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris base</td>
			<td style="width:139px;">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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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。https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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 ...

inducible-lap-tagged-stable-cell-lines-for-investigating-protein

Nanyang Technological University

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

（TET-ON）与四环素诱导shRNA或cDNA表达生成稳定的人类细胞系

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

发现蛋白质相互作用和检定蛋白质功能使用的HaloTag技术

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...