这项研究是由威康信托高级奖学金颁发给伊恩·古德费洛博士。

1。代细胞系:pMEP4转/表达<ol><li>与pMEP4表达载体转染细胞和潮霉素B /毫升（罗氏）与100微克的选择，直到所有的模拟转染细胞被杀害。在pMEP4质粒保持episomally所以没有必要隔离特定的克隆。 </li><li> 10μM2 CDCL 16小时治疗，可诱发细胞包含pMEP4向量。应证实前小规模扩增的细胞株的表达和诱发。作为G蛋白域几乎所有种类的抗体结合，可以很容易地检测到的TAP标签蛋白。对于大规模纯化通常为10合流175厘米2的细胞瓶是必需的。这相当于约2 X10的8表 ​​达细胞。 </li></ol> 2。细胞裂解液的准备<ol><li>刮至PBS的细胞，并结合成一个单一的50毫升管。旋转1200X GF或5分钟（这应该导致在2-3毫升包装细胞开始，清洗后减少到约150毫升）。 </li><li>在冰冷的PBS（每次50毫升）洗细胞三次。 </li><li>在5毫升裂解液（裂解细胞50毫米的Tris-HCl（pH值7.5），125 mM氯化钠，5％甘油，0.2％的NP-40，MgCl 2的 1.5毫米，25毫米，1毫米氟化钠钠4 3旁白和蛋白酶抑制剂）。注:氟化钠，钠3 VO 4和蛋白酶抑制剂应补充新鲜。吸取了前5分钟在冰上留下的10倍。离开冰再次在冰上5分钟前使用钝针注射器上升和下降的5-10倍。重复syringing和离开进一步冰上5分钟。 </li><li>冻融裂解两次（液体N 2 /或干冰块和乙醇）。不要让样品达到大于4°C。注意:您可以储存在-80°C，直到你有时间来处理这个范例。 </li><li>分装到1.5毫升管样本和删除unlysed细胞和Debris通过离心（在4°C，16,000 XĞ10分钟）。 </li><li>恢复上清液，在15毫升猎鹰管相结合，并通过0.45微米的过滤（可选）前通过采取定量蛋白质产量的20μL样品。卸下进一步的后续分析（样品1）50μL等份。 </li></ol> 3。结合兔IgG-琼脂糖 （注:应进行所有的自旋1200XĞ在冷冻离心机1分钟，在4°C，除非另有说明） <ol><li>纷飞瓶轻轻悬浮兔IgG-琼脂糖溶液（Sigma-Aldrich公司）。取380μL的IgG琼脂糖（截止1毫升枪头）和离心1分钟，除去航运/防腐剂的解决方案。琼脂糖三次洗冷冻裂解液（4℃）使用离心澄清。琼脂糖的最终产量应该装珠约250μL。 </li><li>从2.6（15毫升猎鹰浴缸中加入清除的细胞裂解液E）水洗兔IgG琼脂糖孵育3小时（或隔夜）在4°C使用一个旋转的搅拌机。 </li><li>降速琼脂糖珠在4°C 5分钟，取出供后续分析（样品2）上清。注:标签蛋白的TAP现在应与珠。 </li></ol> 4。 TEV蛋白酶切割 （注:应进行所有的自旋1200XĞ在冷冻离心机在4℃1分钟，除非另有说明） <ol><li>冷冻裂解液洗兔IgG琼脂糖珠三次（4°C）使用离心澄清（注:裂解液中不应该包含蛋白酶抑制剂）。应小心，不要删除或失去任何珠在洗涤步骤。 </li><li>洗珠TEV蛋白酶裂解缓冲使用离心澄清（10毫米的Tris-HCl（pH值7.5），100 mM氯化钠，0.2％NP-40）进一步两次。最后一次洗涤后小心取出所有李从珠嚼食。 </li><li>准备一个TEV蛋白酶切割组合;每个样品包括467.5μL 氢澳，25μL20X，TEV缓冲（Invitrogen公司），5微升0.1M数码地面电视和2.5μL（25美）TEV蛋白酶（Invitrogen公司）。加入500μl这个TEV酶切组合的每个样品包装珠和整个组合转移到1.7毫升预润滑管（COSTAR）。之前，以孵化为后续分析（样品3）删除30μL等份。 </li><li> TEV蛋白酶反应过夜孵育4℃用一个旋转的搅拌机。请注意，可用于较短的孵化时间取决于诱饵蛋白和无障碍TEV蛋白酶裂解位点，但最小的孵化时间的性质，应凭经验确定。 </li></ol> 5。结合ULTRALINK固定的链霉加珠 （注:应进行所有的自旋1200XĞ在冷冻离心机1分钟，在4°C，除非另有说明） </P&gt; <ol><li>离心TEV酶切反应，其中包含为1200X克（4℃）5分钟的兔IgG琼脂糖珠。删除的分析澄清上清20μL等份（样品4） </li><li>其余上清液（约480μL）转移到一个新的1.5毫升预润滑管（COSTAR）离开冰。不要扔掉此上清 - 这包含TEV切割诱饵蛋白和任何关联的结合蛋白。 </li><li>新增500个冷藏裂解缓冲液（2.3节），其余兔IgG琼脂糖珠和重悬。这种混合物离心澄清之前，去除上清相结合，从步骤5.2珠。离开冰管（它现在应该包含〜980μL的样品）。 </li><li>保留供后续分析兔IgG琼脂糖珠（样品5）。注:如果你运行这个样本上的SDS-PAGE胶（或印迹），它会给很多背景，由于存在的IgG重型和轻型链。 </li><li>同时轻轻悬浮ULTRALINK固定纷飞的瓶子，加链霉亲和素琼脂糖珠（皮尔斯）。使用切成末，预润滑1.5毫升管分装成70μL链亲和素珠每样本200μL枪头和离心除去运输/防腐剂的解决方案。注意:这些珠子都非常小，所以"鸭子嘴"或平/窄端的技巧可以使用，以尽量减少损失洗涤过程中的珠。 </li><li>洗链亲和素珠冰鲜裂解液3次（4°C）使用离心澄清。这应该留下约45-50微升，每管装珠。 </li><li>转让TEV蛋白酶裂解上清（即从步骤5.3样品980μL）管含3小时（或隔夜）洗链亲和素磁珠孵育4℃用一个旋转的搅拌机。 </li><li>降速在4°C 5分钟的链亲和素珠，去除上清，为后续的分析（山姆PLE 6）。洗链亲和素珠冰鲜裂解液3次（4°C）使用离心澄清。最后一次洗涤后，小心地取出所有剩余的洗涤缓冲液。 </li></ol> 6。素的链亲和素的结合肽和诱饵蛋白的洗脱 （注:应进行所有的自旋1200XĞ在冷冻离心机1分钟，在4°C，除非另有说明） <ol><li>加入500μL的PBS洗脱标记的链亲和素珠蛋白生物素（1毫米D-生物素），并在4°C孵育3小时，用一个旋转的搅拌机（或过夜）。 </li><li>降速5分钟，在4°C的链亲和素珠，并取出上清液1.5毫升预润滑管（注:这是你最后的样品/洗脱（7）样品。 </li><li>另外500μL生物素:PBS加入剩余的链亲和素珠，并在4°C孵育2-3小时，用一个旋转的搅拌机（或过夜）。 </li><li>重新泥炭加强6.2，并结合两个洗脱（样本7）。这是最后的洗脱可以储存在-80°C </li><li>为了评估生物素洗脱效率，冻结余下的链亲和素珠，煮沸后在SDS样品缓冲液（样品8）（建议:减少从费舍尔LaneMarker 5倍样品缓冲液）。 </li></ol> 7。蛋白质浓度<ol><li>最后洗脱液（样品）需要分析前要集中，因为低蛋白浓度相对高容量。这可以使用低分子量（&lt;5 KDA）Vivaspin自旋列（Vivascience）。旨在集中量小于100μL。这最后的样本，可煮，蛋白上样缓冲液中（建议:LaneMarker 5倍减少从费舍尔的样品缓冲液）。 </li></ol> 8。分析<ol><li>样品1-8可以通过免疫印迹分析，以确定下拉效率。注意:大多数中学抗体与G蛋白域的融合蛋白发生交叉反应，但这是TEV蛋白酶裂解后删除。 </li><li>可以1D或2D SDS-PAGE电泳分析样品7。可以使用银或考马斯（建议:SilverQuest银染色和蓝色胶体考马斯染色试剂盒自Invitrogen）进行染色。可以用质谱进行蛋白质鉴定。 </li></ol> 9。代表结果 1D的SDS-PAGE分析，从该协议的最终洗脱（样本7）确定的TAP标签eIF4E的结合伙伴的一个例子是在图2。这代表凝胶反映的复杂和丰富的自然与其他蛋白质在细胞中eIF4E的相互作用。在图2所示的阴性对照，从一个细胞只表达TAP标签生成的比较，说明这eIF4E的诱饵下拉的特殊性。在这种情况下集中的最终洗脱15％（样本7）使用Invitrogen公司从Silverquest银染色试剂盒染色前1D使用市售的预制梯度凝胶的SDS-PAGE分析。 通常50-85％，其余集中的最终洗脱（样本7）进行了分析与胶体考马斯染色（Invitrogen公司）。从整个车道的样品（凝胶片/条），然后提取和分析，质谱分析。 eIF4E的下拉鉴定的蛋白质进行过滤对从阴性对照，以确定非特异性结合合作伙伴的具有约束力的合作伙伴。确定最终的蛋白质使用这个TAP标签eIF4E的过程可以看出，在图2中，这是正常的蛋白质确定使用这种技术的代表。此外，还确定了一些eIF3亚基（数据未显示）。 <img src="/files/ftp_upload/3643/3643fig1.jpg" alt="图1" /> 图1。示意图串联亲和纯化过程。六步串联亲和纯化（TAP），协议涉及细胞的生成，细胞裂解，兔IgG琼脂糖凝胶免疫沉淀，TEV蛋白酶裂解，链霉亲和素磁珠亲和纯化，并终于素洗脱。 <img src="/files/ftp_upload/3643/3643fig2.jpg" alt="图2" /> 图2。串联亲和纯化的小鼠eIF4E的蛋白质。互动合作伙伴的N末端 ​​抽头标签eIF4E的使用所附的协议，从真核细胞转染HEK293细胞进行纯化。一个20％的分数，最后洗脱（样本7），通过SDS-PAGE分析，预铸的4-12％梯度凝胶电泳的TAP-eIF4E的车道上。一个单独的标签（TAP车道）进行等效分析。用银染蛋白。随后通过对同一样品质量法确定的蛋白质强调这种凝胶。 缩写:eIF4G;真核翻译起始f演员4伽马，PABP认证;多聚腺苷酸结合蛋白，eIF4A:真核翻译起始因子4α，收缩压，eIF4E的;其余的TAP诱饵蛋白，含有融合的真核翻译起始因子4E，4EBPs;真核生物翻译起始因子4E结合的链亲和素的结合肽蛋白质，eIF4eNiF1l真核翻译起始因子4E核进口因子1，收缩压（SBP），其余的从TEV非稠合的TAP肽链霉结合肽。

<ol>
	<li>Williamson, M. P., Sutcliffe, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions.">Protein-protein interactions.</a> Biochem. Soc. Trans. 38 (4), 875-878 (2010).</li><li>Rigaut, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat. Biotechnol. 17 (10), 1030-1032 (1999).</li><li>Blackwell, C., Brown, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+tandem-affinity+purification+to+Candida+albicans.">The application of tandem-affinity purification to Candida albicans.</a> Methods Mol. Biol. 499, 133-148 (2009).</li><li>Cochrane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+complex+formation+between+HIV+Rev+and+the+nucleosome+assembly+protein,+NAP1,+affects+Rev+function.">Stable complex formation between HIV Rev and the nucleosome assembly protein, NAP1, affects Rev function.</a> Virology. 388 (1), 103-111 (2009).</li><li>Fernandez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+tandem+affinity+purification+of+PSD-95+recovers+core+postsynaptic+complexes+and+schizophrenia+susceptibility+proteins.">Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.</a> Mol. Syst. Biol. 5, 269-269 (2009).</li><li>Gloeckner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+of+protein+complexes+from+mammalian+cells+by+the+Strep&#47;FLAG+(SF)-TAP+tag.">Tandem affinity purification of protein complexes from mammalian cells by the Strep&#47;FLAG (SF)-TAP tag.</a> Methods Mol. Biol. 564, 359-372 (2009).</li><li>Holowaty, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+profiling+with+Epstein-Barr+nuclear+antigen-1+reveals+an+interaction+with+the+herpesvirus-associated+ubiquitin-specific+protease+HAUSP/USP7.">Protein profiling with Epstein-Barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease HAUSP/USP7.</a> J. Biol. Chem. 278 (32), 29987-29994 (2003).</li><li>Burckstummer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+tandem+affinity+purification+procedure+for+interaction+proteomics+in+mammalian+cells.">An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.</a> Nat. Methods. 3 (12), 1013-1019 (2006).</li><li>Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calicivirus+translation+initiation+requires+an+interaction+between+VPg+and+eIF+4+E.">Calicivirus translation initiation requires an interaction between VPg and eIF 4 E.</a> EMBO Rep. 6 (10), 968-972 (2005).</li><li>Goodfellow, I. G., Roberts, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+initiation+factor+4E.">Eukaryotic initiation factor 4E.</a> Int. J. Biochem. Cell Biol. 40 (12), 2675-2680 (2008).</li></ol>

没有利益冲突的声明。

说明这里的TAP标签技术演示为诱饵蛋白在真核细胞中分离的具有约束力的合作伙伴的高度特异性和严格的方法。这种方法可应用于细胞和病毒蛋白。据我们所知，这是第一次，这种技术已被应用到翻译起始因子eIF4E。知名eIF4E的结合蛋白eIF4G和使用这种技术的4EBPs鉴定，证实了这种方法的有效性。此外，鉴定eIF4F复杂的剩余部分，​​即eIF4A和PABP至证实，间接的互动关系和大专院校的复合物留在净化过程中的完整。 eIF4E的结合蛋白的典型多个亚型的识别也很明显。图2中更详细地描述了这些。 至于该方法的局限性，某些保健应采取的选择与问候诱饵蛋白和是否放置在N-或C-末端的TAG标签。这可能是最好进行功能检测，或本地化融合蛋白通过显微镜检查，以确保标签的衍生工具是功能完整的大规模纯化。积分膜或核蛋白质不一定被释放裂解步骤中所述的条件相对温和的洗涤剂。与所有免疫沉淀和类似拉下来分析，修改，可以在提高效率的裂解裂解液的性质和浓度的洗涤剂。离子浓度的溶解和洗涤缓冲也可以修改，增加或减少净化的严格。它也可以执行初步净化温和条件下标准（如上所述），但随后分裂的链亲和素珠（5.8节）为4-5等份，随后被冲下离子浓度增加ditions。这可能使用户能够确定的最佳条件，消除非特异性相互作用的蛋白质。整个过程中也可以使用互动的合作伙伴被称为瞬时转染和随后印迹确定其存在或不存在。在这种情况下，我们建议各8微克的表达质粒转染细胞100厘米2菜。这个修改​​过程是研究突变衍生物的TAP融合蛋白与已知的具有约束力的合作伙伴互动的能力时，特定用途。 分析样品1，通过银染SDS-PAGE凝胶（或印迹，如果抗体是可用的）8，是一个很好的方法排除故障，技术故障产生可接受的结果。使用此系统抽样的方法，可以分析的基于每个亲和力的降水和具体洗脱效率。样品应采取一至八个期间OPTIM化的协议，为每个新的诱饵。 TAP标签技术提供了一个强大和稳健的替代其他方法，如GST拉，酵母双杂交实验等双亲和纯化和具体洗脱步骤（TEV酶切和生物素洗脱）提供特异性和严格保持在整个净化过程中的较高水平。批判这种技术可以应用到任何蛋白质，因此确定为目标蛋白结合合作伙伴的一个极好的方法。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>潮霉素B </td><td>罗氏公司</td><td> 10843555001 </td></tr><tr><td>兔IgG的琼脂糖</td><td>西格玛</td><td> A2909 </td></tr><tr><td> AC-TEV蛋白酶</td><td> Invitrogen公司</td><td> 12575-015 </td></tr><tr><td>蛋白酶抑制剂</td><td> Calbiochem公司</td><td> 539134 </td></tr><tr><td> ULTRALINK链霉亲和素固定加珠</td><td>刺穿</td><td> 53116 </td></tr><tr><td> vivaspin 500离心选矿（5KDa） </td><td> vivaspin </td><td> V50112 </td></tr><tr><td> SilverQuest银染试剂盒</td><td> Invitrogen公司</td><td> LC6070 </td></tr><tr><td> NOVEX蓝色胶体考马斯染色试剂盒</td><td> Invitrogen公司</td><td> LC6025 </td></tr><tr><td> 1.7毫升预润滑管</td><td> COSTAR </td><td> 3207 </td></tr><tr><td>微毛细管移液器提示</td><td>厂商VWR </td><td> 37001-150 </td></tr><tr><td> NuPage 4-12％二，三梯度凝胶</td><td> Invitrogen公司</td><td> NP0322BOX </td></tr><tr><td> CDCL 2 </td><td>西格玛</td><td> 202908 </td></tr><tr><td> 5X SDS样品缓冲</td><td>费舍尔</td><td> PN39000 </td></tr></tbody></table>

identification of protein interacting partners using tandem affinity purification

一个经常限制的关键一步，在了解主机和病毒蛋白的功能是相互作用的细胞或病毒蛋白伙伴的标识。有许多方法，使识别的互动合作伙伴，包括酵母双杂交系统，以及下拉检测用重组蛋白免疫沉淀和内源性蛋白质质谱鉴定1。最近的研究都强调实用的双亲和力标签介导的净化，再加上两个相互作用的蛋白质鉴定的具体洗脱步骤。这种方法称为串联亲和纯化（TAP）的，最初用于酵母2,3，但最近已经适应在哺乳动物细胞中的使用4-8。 作为证据的概念，我们已经建立了串联亲和纯化（TAP）的方法，利用良好的特点真核翻译起始因子TOR eIF4E的9,10。细胞翻译因子eIF4E是一个蜂窝eIF4F复杂的重要组成部分，涉及帽依赖的翻译起始10。在目前的研究中使用的TAP标签是两个G蛋白单位组成，由烟草蚀纹病毒（TEV）蛋白酶切割序列1链亲和素结合肽的分离。在目前的研究中使用的TAP标签组成的两个G蛋白单位和1链亲和素结合肽的分离，由烟草蚀纹病毒（TEV）蛋白酶切割序列8。放弃代克隆细胞系的需要，我们开发了一种快速的系统依赖于从episomally保持质粒的TAP标签的诱饵在pMEP4（Invitrogen公司）为基础蛋白表达。使用氯化镉诱导金属硫蛋白启动子控制的标签从这个质粒的小鼠eIF4E的表达。 通过结合到R的表达和随后的亲和纯化的裂解abbit抗体琼脂糖，TEV蛋白酶裂解，结合链霉联系琼脂糖和生物素洗脱随后确定了许多蛋白质​​显然是具体的eIF4E的下拉（时相比，控制细胞株单独表达TAP标签）。蛋白质的身份获得了从一维的SDS-PAGE和随后的串联质谱带切除。确定的组件包括知名eIF4E的结合蛋白eIF4G和4EBP-1。此外，eIF4F复杂的其他组成部分，其中eIF4E的是一个组件被确定，即eIF4A和聚A结合蛋白。识别能力，不仅直接约束力的合作伙伴以及二级相互作用的蛋白质，进一步突出了在未知功能的蛋白质的特性，这种方法的效用。

Watch this Scientific Journal Video about Identification of Protein Interacting Partners Using Tandem Affinity Purification at JoVE.com

Identification of Protein Interacting Partners Using Tandem Affinity Purification

串联亲和纯化的蛋白结合合作伙伴的标识是稳健的做法。由于概念证明，这种方法的应用以及特色的翻译起始因子eIF4E共沉淀的宿主细胞因子参与翻译起始。这种方法是很容易适应任何细胞或病毒蛋白。

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

identification-protein-interacting-partners-using-tandem-affinity

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

37.4K 次观看.  Imperial College London. 串联亲和纯化的蛋白结合合作伙伴的标识是稳健的做法。由于概念证明，这种方法的应用以及特色的翻译起始因子eIF4E共沉淀的宿主细胞因子参与翻译起始。这种方法是很容易适应任何细胞或病毒蛋白。

视频: Identification of Protein Interacting Partners Using Tandem Affinity Purification

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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

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

从真核细胞蛋白质复合体串联亲和纯化

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

科研

生物化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

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

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

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

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

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

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

生物学

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

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 ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

本机配合物的净化对结构研究使用串联亲和标签法

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

用于研究蛋白质-蛋白质相互作用的串联亲和纯化检测

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

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