The authors are grateful for the support and advice of Nikolaus Grigorieff. We thank Anna Loveland, Axel Brilot, Chen Xu, and Mike Rigney for helpful discussions and EM guidance. This work was funded by the National Science Foundation, Award No. 1157892. The Brandeis EM facility is supported by National Institutes of Health grant P01 GM62580.

注意:下面的协议被设计为一个复杂的纯化从4升细胞培养物，细胞的大约40克湿重。一旦制备，所有缓冲区应储存在4℃并且在一个月它们的制备中使用。还原剂和蛋白酶抑制剂仅加入缓冲液在使用前。 1.全细胞提取的制备串联亲和纯化<ol><li> S.生长酵母细胞 <ol><li>连胜所需的TAP标记S.酵母菌株从存储器（-80°C）到酵母蛋白胨葡萄糖（YPD）板。孵育3 - 5天（30°C），直到酵母细胞出现白色蓬松。 </li><li>接种约2mm从板的新鲜细胞的2条纹到10ml的YPD培养基的50ml锥形聚丙烯离心管中。摇动该管以每分钟180转（rpm）过夜（30米℃）。 </li><li>接种将2ml超过每升YPD媒体的夜间文化在2升锥形或锥形瓶中。摇匀，以180rpm（30°C）。监测在600nm（OD 600）的光密度的细胞生长，直至晚对数期，1.8的OD 600为2时，典型地为20 - 24小时。 </li></ol></li><li> 收获S.酵母细胞 <ol><li>收获细胞，离心5,400×g离心15分钟（4℃）。 </li><li>迅速倒出上清液，并保持细胞在4℃或冰上。 </li><li>暂停用2ml冷却裂解缓冲液每升细胞沉淀（10毫摩尔Tris-HCL，pH8.0中; 300mM NaCl的10毫米氯化钾; 0.2毫摩尔EDTA，pH值8.0; 5mM咪唑，pH值8.0; 10％甘油; 0.1％体积/体积的NP-40）。通过涡旋和/或使用10ml的血清吸管重复移液悬浮细胞。 注意:NP-40的重要性，因为它涉及到复杂的质量和纯化后的分析在下面讨论。 </li><li>细胞悬液转移到一个空的50ml锥形聚丙烯离心管组合EPT冰上。洗每个离心瓶中用另外2毫升冷却裂解缓冲液，并加入到细胞悬浮液中的50ml管中。 </li><li>通过称量管和减去空管的重量以及增加的裂解缓冲液确定细胞沉淀的湿重。 注:升具有外径为1.8 600的细胞培养的约为10克</li><li>创建在50ml锥形聚丙烯离心管中的液态氮浴中。绘制悬浮的细胞加到5毫升注射器。一个地下16针连接到注射器。使其通过注射器/针头以产生细胞冷冻细胞悬浮液"液滴"。冷冻的速率应为约1分钟/毫升。 </li><li>储存冷冻细胞液滴（-80℃），或进行裂解。 </li></ol></li><li> 裂解细胞和裂解液澄清 <ol><li>裂解用咖啡研磨机的酵母细胞。裂解细胞八至九倍25秒磨阵阵，一阵晃动的C奥菲磨床。在使用前，预冷却用液氮的咖啡研磨机。每两轮研磨，加液氮浅层的磨床，并允许它蒸发。 </li><li>以维持细胞凝集用液氮冷却的刮刀搅拌。一定要小心，防止细胞，这可能会导致细胞聚集的解冻。细胞应出现如下裂解细的白色粉末。来自4 L酵母培养物（〜40克）的最大细胞沉淀可以同时被粉碎。 注意:关于裂解的方法观察在下面讨论。 </li><li>评估细胞裂解使用血球或替代细胞计数法的程度。为了提供足够的分析，采取以下样品:（1）裂解之前; （2）经过四轮裂解;和（3）下列裂解。检查这些样品，取细胞的少量用在1.5ml管中的液态氮冷却的刮刀和地点。 </li><li>一旦细胞被解冻，吸管5微升细胞并用495微升水混合。如果40 - 观察细胞的60％溶解，继续在该协议下一个步骤。 注意:对于裂解的长时间的影响观测在下面讨论。 </li><li>一旦足够的裂解观察到，卖场裂解细胞（-80℃）或进行下一步。 </li><li>制备两个50毫升锥形聚丙烯离心各用15毫升裂解液管，其中1毫苯甲基磺酰氟（PMSF），1mM的二硫苏糖醇（DTT），和蛋白酶抑制剂的市售的鸡尾酒。 注意:如果发现显著蛋白水解，可考虑使用蛋白酶缺陷菌株。 </li><li>使用液态氮冷冻铲冷冻裂解细胞粉舀入准备好的50ml试管。逐步增加的细胞粉/固体的裂解液，含有还原剂和蛋白酶抑制剂（S）。 </li><li>旋转50ml试管轻柔地在RT以便解冻/溶解的细胞，也能防止气泡的形成。一个S中的细胞粉/固体溶解，增加额外的电池固体/粉末。 </li><li>填写每两个50毫升管，直到每个人都有大约20g细胞或添加所有单元格。继续解冻/溶解，直到有在50ml试管没有观察到冷冻的细胞团块。这大约需要50分钟。 </li><li>离心20分钟，悬浮细胞裂解物以25,000rpm XG（4℃）。良好的溶解的细胞可表现出黑色沉淀物的量小。 </li><li>转移50ml的上清液以聚碳酸酯超离心管（用于26.3毫升管），并加入10微升200毫米PMSF每个全管。 </li><li>离心以100,000 xg离心（4℃）1小时。四层应该是可见的，从底部到顶部:（1）硬颗粒清晰，含有核糖体复合物; （2）软富含脂质的沉淀; （3）含有大部分细胞的可溶性蛋白和复合物的大透明黄色色彩的层;和（4）&#39;鳞屑"顶层组成的脂质。 </li><li>执行所有特殊在冷室（4℃）ubsequent步骤。使用1毫升移液管除去尽可能多的顶部的"鳞片状"脂质层尽可能的并丢弃。用10毫升血清吸管收回大部分黄色，层次清晰的。 </li><li>使用1毫升吸移管以避免扰乱底部两层恢复的最后几毫升该层。由40克湿细胞沉淀，约48毫升中间层的典型回收和8毫升脂质层的去除/丢弃。 注:柱纯化流由脂质，其也可以降低该复合物的质量的存在极大地阻碍了，在下面讨论。 </li></ol></li></ol> 2.柱纯化步骤1:IgG的色谱<ol><li> 树脂平衡和绑定 <ol><li>准备一个300微升的IgG琼脂糖浆40 G细胞颗粒。剪下1毫升枪头年底，以方便移液浆料。洗涤/平衡IgG的树脂三次，每次用5ml IgGD150 BuffeR（10毫摩尔Tris-HCL，pH8.0中; 150毫摩尔NaCl; 150毫氯化钾; 1mM的MgCl 2的; 5mM咪唑，pH值8; 0.1％体积/体积的NP-40; 1毫摩尔DTT;每50蛋白酶抑制剂片剂毫升体积）。 <ol><li>旋在160的洗涤之间XG（4℃）沉淀并除去上清。 </li></ol></li><li>旋转的IgG树脂与中间相上清液，每40 G细胞两个迷你蛋白酶抑制剂的片剂，使用适当的旋转器，2小时（4℃）。这是在IgG的批量溶液。 </li><li>通过切割色谱柱末端产生平砍准备用两个10 ml聚预习列。为了加快通过重力流柱的填料和洗涤，装入一个最大的打包的IgG树脂的200微升或约25mL每10 ml柱IgG的批量溶液。 </li><li>倾IgG的批量溶液进入塔，并允许通过重力沉降。如果沉降超过30分钟的时间较长，有回收从离心机T中的中间阶段时，最有可能是脂质污染ubes。 </li><li>有四个连续10毫升卷IgGD150缓冲液洗填充柱。 </li></ol></li><li> 在列TEV切割 <ol><li>密封柱的底部并加入1毫升IgGD150缓冲液和100微升TEV蛋白酶的（0.6毫克/毫升的库存，TEV的突变体变体S219V产生的内部）。密封该塔的顶部并混合使用热混合器中，在750rpm下摇动20分钟（18℃）。悬浮树脂和再次混合20分钟，重复一次，共1小时温育。 注意:这是关键的，以保持温育的时间为最小。 </li><li>返回列至4℃并通过重力洗脱蛋白/复合物。洗脱死体积增加200微升IgGD150缓冲。 </li></ol></li></ol> 3.柱纯化步骤2:钙调蛋白亲和层析<ol><li> 树脂平衡和绑定 <ol><li>制备每40 G细胞的200μl的钙调素亲和树脂的沉淀。洗树脂三次，每次用5ml钙调蛋白结合缓冲液（10mM的Tris-HCl，pH值8.0; 150毫摩尔NaCl; 10毫氯化钾; 1mM的MgCl 2的; 5mM咪唑，pH值8; 2毫米氯化钙2; 0.1 ％体积/体积的NP-40; 10毫β巯基乙醇）中，在160 xg离心（4℃）离心以沉淀每次洗涤。 </li><li>为了将每1ml的IgG洗脱液，加入3卷钙调素结合缓冲液。为了保持血钙水平不变，加入氯化钙和β巯基乙醇占缺乏IgG的洗脱这些成分。终浓度应在2毫米氯化钙2和10mMβ巯基乙醇。 </li><li>样品添加到钙调素树脂和绑定使用的管旋转1小时（4℃）。 </li></ol></li><li> 包装和钙调蛋白树脂洗脱 <ol><li>通过切割列的端部创建一扁平开口制备每100μl的钙调素的浆液2ml的聚制备柱。每加载合作不超过100微升钙调蛋白浆液lumn尽量减少树脂样品的量来在洗脱过程中接触。 </li><li>包通过重力柱并三次用5ml钙调素结合缓冲液洗涤树脂在列。 </li><li>洗脱蛋白并加入200微升钙调素的洗脱缓冲液（10mM的Tris-HCl，pH值8.0的150毫摩尔NaCl; 10毫氯化钾; 1mM的MgCl 2的; 5mM咪唑，pH值8.0; 4毫EGTA，pH 8.0的0.08％体积/ v的NP-40; 10毫β巯基乙醇）。重复加入200微升钙调素洗脱缓冲液六次。 </li><li>为洗脱1〜3中，应用量给列和立即收集洗脱液。洗脱4，密封该塔的底部，并与洗脱缓冲液孵育2.5分钟，然后启封，并允许通过重力洗脱。用于洗脱5，孵育5分钟，然后启封和洗脱。用于洗脱6，孵育10分钟，然后启封和洗脱。 注:复杂的完整性可能洗脱液之间变化/分数（见代表性的结果节）&lt;。/ LI&gt; <li>透析洗脱2至6隔夜单独使用合适的微透析法。透析针对透析缓冲液的级分中（10mM的Tris-HCl，pH值8.0; 150毫摩尔NaCl; 10毫氯化钾; 1mM的MgCl 2的; 5mM咪唑，pH值8; 10毫β巯基乙醇）。 注意:NP-40不会明显通过，如果在所有通过透析膜。 </li></ol></li></ol> 4.后净化分析，评估综合素质<ol><li> 天然凝胶银染 <ol><li>制备4 - 16％预制天然凝胶的配合物作为每从制造商的指示进行分析。使用每个透析钙调素的洗脱/级分的合适的分子量蛋白质标准和负载15微升到凝胶。 </li><li> Electrophorese在150V凝胶为约3小时（4℃）。 </li><li>银染色凝胶，以评估各六个洗脱液的质量。另外，使用同样敏感的商业荧光染色（多个）。在这个阶段中，配合物的浓度是最有可能低于检测为考马斯蓝染色的水平。 </li></ol></li><li> 附加凝胶分析方法 <ol><li>为了检测通过蛋白质印迹蛋白，解决通过SDS或变性PAGE复杂，然后探测用的TAP标记抗体作为每从制造商的说明进行钙调蛋白结合肽（CBP）的表位的凝胶。 </li><li>使用特定的荧光染料（S），确认在TAP纯化复杂的存在和蛋白质和/或核酸的完整性。采取这些污物间没有检测到的频谱重叠照顾。 </li></ol></li><li> 负染电子显微镜可视化 <ol><li>使用连续碳网格辉光放电在-15毫安，持续30秒，复杂的应用的30分钟之内。 </li><li> （通常在1nM的浓度）应用3微升的TAP纯化复杂的给电网。孵育一分钟。印迹˚FROM网格的一侧，以除去过量的缓冲。 </li><li>用的去离子水20微升滴洗两次。洗涤之间边印迹。 </li><li>应用网格50微升负染色下降，孵育一分钟。推荐染色醋酸铀（2％）或铀酸盐（0.75％）。 注意:盐铀和解决方案是弱放射性和含有重金属。这些应该谨慎，并附带在这些解决方案应处置以下机构推荐废物准则的接触的材料进行处理。 </li><li>应用网格到一个新的50微升负染下降，无印迹。孵育一分钟，然后一边印迹，最后空气干燥和可视化。 </li></ol></li><li> 低温电子显微镜（冷冻电镜）可视化 <ol><li>与TAP纯化复杂执行冷冻电镜，虽然优化可能是必要的，根据制造商的协议。 注:洗涤剂存在高浓度马Ÿ阻碍进步和下面讨论。 </li></ol></li></ol> 5.复杂的存储<ol><li> 准备对复杂存储 <ol><li>浓缩复合物，如果需要的话，使用具有合适分子量截断的复杂的离心过滤器。在14000 XG 1分钟为增量旋转达到所需的浓度为止。 注意:NP-40浓度将浓缩过程中的浓度增加，因为它不通过过滤器，也不透析膜。 </li><li>闪存冻结在液氮或干冰30微升等分复杂的冷却乙醇浴，然后在-80℃保存。 </li></ol></li><li> 洗涤剂的可选的后去除净化 </li></ol>注意:去污剂例如NP-40，并在高于其临界胶束浓度可能妨碍或抑制对于一些应用进展的浓度存在。其去除从原后果栏以及与其它添加剂或共溶剂替换在下面讨论。如果没有NP-40是需要的，一个方法是使用用于去除一个商业应用，例如生物珠。 <ol style="margin-left: 40px;"><li>在透析缓冲液预均衡市售洗涤剂吸收珠。混合每络合物30微升5珠（通常以10nM浓度）。 </li><li>来自步骤5.2.1在冰上15分钟的TAP纯化复杂孵育平衡珠。除去洗涤剂的以下几个小时之内使用复杂的。 </li></ol>

<ol>
	<li>Gavin, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes.">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415 (6868), 141-147 (2002).</li><li>Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., Seraphin, 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+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nature Biotechnology. 17 (10), 1030-1032 (1999).</li><li>Puig, O., 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+tandem+affinity+purification+(TAP)+method:+A+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: A general procedure of protein complex purification.</a> Methods. 24 (3), 218-229 (2001).</li><li>Vaillancourt, P., Zheng, C. F., Hoang, D. Q., Breister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+recombinant+proteins+fused+to+calmodulin+or+to+calmodulin-binding+peptides.">Affinity purification of recombinant proteins fused to calmodulin or to calmodulin-binding peptides.</a> Methods Enzymol. 326, 340-362 (2000).</li><li>Braisted, A. C., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+a+binding+domain+from+protein+A.">Minimizing a binding domain from protein A.</a> Proc Natl Acad Sci U S A. 93 (12), 5688-5692 (1996).</li><li>Kapust, R. 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=Tobacco+etch+virus+protease:+mechanism+of+autolysis+and+rational+design+of+stable+mutants+with+wild-type+catalytic+proficiency.">Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.</a> Protein Eng. 14 (12), 993-1000 (2001).</li><li>Nallamsetty, S., 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=Efficient+site-specific+processing+of+fusion+proteins+by+tobacco+vein+mottling+virus+protease+in+vivo+and+in+vitro.">Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro.</a> Protein Expr Purif. 38 (1), 108-115 (2004).</li><li>Ghaemmaghami, S., 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=Global+analysis+of+protein+expression+in+yeast.">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li><li>Howson, 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=Construction,+verification+and+experimental+use+of+two+epitope-tagged+collections+of+budding+yeast+strains.">Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains.</a> Comp Funct Genomics. 6 (1-2), 2-16 (2005).</li><li>Cox, D. M., Du, M., Guo, X., Siu, K. W., McDermott, J. C. <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.">Tandem affinity purification of protein complexes from mammalian cells.</a> Biotechniques. 33 (2), 267-268 (2002).</li><li>Gully, D., Moinier, D., Loiseau, L., Bouveret, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+partners+of+acyl+carrier+protein+detected+in+Escherichia+coli+by+tandem+affinity+purification.">New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification.</a> FEBS Lett. 548 (1-3), 90-96 (2003).</li><li>Tharun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+analysis+of+the+decapping+activator+Lsm1p-7p-Pat1p+complex+from+yeast.">Purification and analysis of the decapping activator Lsm1p-7p-Pat1p complex from yeast.</a> Methods Enzymol. 448, 41-55 (2008).</li><li>Xu, X., Song, Y., Li, Y., Chang, J., Zhang, H., An, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+method:+an+efficient+system+for+protein+complex+purification+and+protein+interaction+identification.">The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.</a> Protein Expr Purif. 72 (2), 149-156 (2010).</li><li>Cheng, Y., Grigorieff, N., Penczek, P. A., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Primer+to+Single-Particle+Cryo-Electron+Microscopy.">A Primer to Single-Particle Cryo-Electron Microscopy.</a> Cell. 161 (3), 438-449 (2015).</li><li>Gottschalk, 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=A+comprehensive+biochemical+and+genetic+analysis+of+the+yeast+U1+snRNP+reveals+five+novel+proteins.">A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.</a> RNA. 4 (4), 374-393 (1998).</li><li>Neubauer, G., Gottschalk, A., Fabrizio, P., Seraphin, B., Luhrmann, R., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+proteins+of+the+yeast+U1+small+nuclear+ribonucleoprotein+complex+by+mass+spectrometry.">Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry.</a> Proc Natl Acad Sci U S A. 94 (2), 385-390 (1997).</li><li>Lucast, L. J., Batey, R. T., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+purification+of+a+stable+form+of+recombinant+tobacco+etch+virus+protease.">Large-scale purification of a stable form of recombinant tobacco etch virus protease.</a> Biotechniques. 30 (3), 544-546 (2001).</li></ol>

The authors have nothing to disclose.

该TAP方法利用两个标签来平衡需要严格和选择性的结合到亲和树脂保持接近生理溶液条件的愿望。这种平衡的作用是保持与净化后的特征相互作用的因子（S）标签蛋白的稳定的相互作用（S）。此外，个别的TAP标记的ORFs可从商业来源，这样就可以得到具有为任何酵母复杂的标签化蛋白质的任何酵母菌株。保留的复合物的完整性和资源，以测试在用于纯化的复杂使用不同标记的蛋白质亚基的可用性?...

许多主要的细胞过程是由大的蛋白质和蛋白质-RNA复合物1进行。一个显著瓶颈进行这种复合物的生物物理和结构研究是获得并在适当浓度的合适的质量（ 即 ，均匀性）它们。从天然来源隔离一个复杂的具有许多优点，包括护亚单位的相关转录后和/或翻译后修饰和投保适当的复杂的装配。然而，大蜂窝络合物以低拷贝数通常存在于细胞和纯化必须高效和近生理条件下发生，以确保复杂的完整性。来自真核来源纯化络合物是特别具有挑战性的，并且可以在经济上令人望而却步。因此，是有效率和产生均匀复杂的策略或方法是高度期望的。 这种策略已经成功地从净化真核细胞的天然复合物为他们的初步定性是串联亲和纯化（TAP）的方法2,3。该方法TAP最初设计在复杂的纯化由芽殖酵母（ 酿酒酵母）天然蛋白与之交互因子（S）2。该方法TAP利用了两个标签，串联融合到同一个蛋白质编码基因序列的每个标签。被选择的代码，以便平衡需要紧和选择性结合的亲和树脂，以保持接近生理溶液条件的愿望。这种平衡的作用是保持与净化后的特征相互作用的因子（S）标签蛋白的稳定的相互作用（S）。的基因组引入的TAP标记被放置在末端（C-末端）的蛋白质编码基因的和由一个序列编码的钙调蛋白结合肽（CBP），随后通过蛋白A的 - 加成刚刚超过20千道尔顿到标签蛋白。 CBP短，26个氨基酸，并通过识别的〜17 kDa蛋白钙调素在钙的存在下用K 的D 10 -9量级M 4上。蛋白A标记是较大，由58残两个重复与重复之间的短连接体。每58个氨基酸重复由免疫球蛋白G（IgG）的用K D〜10 -8 M 5识别。这两个标记之间成立的TEV蛋白酶，从烟草蚀纹病毒6,7内肽酶的识别位点。 如图1所示，在TAP标记蛋白的方法的第1亲和性步骤经由蛋白A的标记的蛋白质是由在加入TEV蛋白酶，位点特异性之间切割的洗脱上柱裂解绑定至IgG树脂这两个标签。这是一个必要的步骤为IgG的互动和蛋白A是非常强的，只能解变性的条件下得到充分的扰动。由于缺乏公关otein一个标记，该蛋白质结合到CaM的树脂在钙的存在下，并从该树脂与另外的金属离子螯合剂EGTA（乙二醇四乙酸）（ 图1）的洗脱。 引进TAP方法，它在一个大规模研究用于生成不久后，"地图"在S.复杂的相互作用酵母 8。重要的是，作为这种努力的整个酵母-TAP标记开放阅读框（ORF）库的结果，以及个别的TAP标记的ORF 9是可从商业来源。因此，可以得到具有为任何酵母复杂的标签化蛋白质的任何酵母菌株。在TAP方法还刺激了修改或在TAP标记的变体，其中包括:其用于从其它真核以及细菌细胞10,11配合物的纯化使用;一个"拆分标签"，其中蛋白质A和CBP被放置在不同的蛋白质12的设计;和TAGS改变，以便容纳研究者的需要，如复合物的 Ca 2+或EGTA 13的灵敏度。 在这两个仪器和方法学的最新进展已经导致了结构确定在电子显微镜（EM）的应用显著的进步，已导致高，接近大分子复合14的原子分辨率的图像。通过获得EM复杂的分辨率，但是，仍然在研究中的复杂的质量而定。这项研究利用了TAP标签的方式从S.净化酵母 U1的核蛋白，18亚基（〜0.8 MDA）低拷贝数核糖核蛋白复合物，是剪接体15,16的一部分。一些已采取步骤以纯化这种复合物，例如，它是均匀的和适当的浓度。在纯化的各阶段中遇到的潜在问题被描述，并采取克服CHALL策略恩格斯强调。通过仔细评估和净化优化步骤中，U1核蛋白纯化是适用于负染色和电子显微镜冷冻（冷冻电镜）研究数量质量和。用于结构研究天然复合物的纯化的优化TAP方法协议在本文中描述。

<table><tbody><tr><td>S. cerevisiae TAP tagged strain</td><td>Open Biosystems</td><td>YSC1177</td><td>This is the primary yeast strain used to develop the TAP protocol. Its background is S288C: ATCC 201388: MATa, his3&amp;Delta;1, leu2&amp;Delta;0, met15&amp;Delta;0, ura3&amp;Delta;0, SNU71::TAP::HIS3MX6</td></tr><tr><td>Coffee grinder</td><td>Mr. Coffee</td><td>IDS77</td><td>Used for cell lysis</td></tr><tr><td>Hemocytometer, Bright Line</td><td>Hausser Scientific</td><td>3120</td><td>Used to assess cell lysis</td></tr><tr><td>JA 9.100 centrifuge rotor&amp;nbsp;</td><td>Beckman Coulter, Inc.</td><td></td><td>Used to harvest the yeast cells</td></tr><tr><td>JA 20 fixed-angle centrifuge rotor</td><td>Beckman Coulter, Inc.</td><td></td><td>Used to clear the cell extract of non-soluble cellular material</td></tr><tr><td>Ti 60 fixed-angle centrifuge rotor</td><td>Beckman Coulter, Inc.</td><td></td><td>Used to further clear the soluble cell extract</td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>R5355</td><td>Temperature controlled shaker</td></tr><tr><td>Novex gel system</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td></td><td></td></tr><tr><td>IgG resin</td><td>GE Healthcare</td><td>17-0969-01</td><td>Sepharose 6 fast flow</td></tr><tr><td>Calmodulin resin</td><td>Agilent Technologies, Inc.</td><td>214303</td><td>Affinity resin</td></tr><tr><td>Protease inhibitor cocktail, mini tablets</td><td>Sigma Aldrich</td><td>589297</td><td>Mini cOmplete ultra EDTA-free tablets</td></tr><tr><td>Protease inhibitor cocktail, large tablets</td><td>Sigma Aldrich</td><td>5892953</td><td>cOmplete ultra EDTA-free tablets</td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td></td><td></td><td>Dissolved in isopropanol</td></tr><tr><td>2 ml Bio-spin column</td><td>Bio-Rad Laboratories, Inc.</td><td>7326008</td><td>Used to pack and wash the Calmodulin resin</td></tr><tr><td>10 ml poly-prep column</td><td>Bio-Rad Laboratories, Inc.</td><td>7311550</td><td>Used to pack and wash the IgG resin</td></tr><tr><td>Precast native PAGE Bis-Tris gels</td><td>Life Technologies</td><td>BN1002</td><td>Novex NativePAGE Bis-Tris 4 - 16% precast polyacrylamide gels</td></tr><tr><td>NativeMark protein standard</td><td>Thermo Fisher Scientific</td><td>LC0725</td><td>Unstained protein standard used for native PAGE. Load 7.5 &amp;mu;l for a silver stained gel and 5 &amp;mu;l for a SYPRO Ruby stained gel</td></tr><tr><td>Precast SDS PAGE Bis-Tris gels</td><td>Life Technologies</td><td>NP0321</td><td>Novex Nu-PAGE Bis-Tris 4 - 12% precast polyacrylamide gels&amp;nbsp;</td></tr><tr><td>PageRuler protein standard</td><td>Thermo Fisher Scientific</td><td>26614</td><td>Unstained protein standard used for Western blotting</td></tr><tr><td>SDS running buffer</td><td>Life Technologies</td><td>NP0001</td><td>1x NuPAGE MOPS SDS Buffer</td></tr><tr><td>TAP antibody</td><td>Thermo Fisher Scientific</td><td>CAB1001</td><td>Primary antibody against CBP tag</td></tr><tr><td>Secondary antibody</td><td>Thermo Fisher Scientific</td><td>31341</td><td>Goat anti-rabbit alkaline phosphatase conjugated</td></tr><tr><td>BCIP/NBT</td><td>Thermo Fisher Scientific</td><td>34042</td><td>5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium&amp;nbsp;</td></tr><tr><td>Dialaysis units</td><td>Thermo Fisher Scientific</td><td>88401</td><td>Slide-A-Lyzer mini dialysis units</td></tr><tr><td>Centrifugal filter units, 100kDa MWCO</td><td>EMD Millipore</td><td>UFC5100008</td><td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td></tr><tr><td>Detergent absorbing beads</td><td>Bio-Rad Laboratories, Inc.</td><td>1523920</td><td>Bio-bead SM-2 absorbants</td></tr><tr><td>SYBR Green II</td><td>Thermo Fisher Scientific</td><td>S-7564</td><td>Flourescent dye for nucleic acid staining, when detecting with SYPRO Ruby present, use excitation wavelength of 488 nm and emission wavelength of 532 nm</td></tr><tr><td>SYPRO Ruby</td><td>Molecular Probes</td><td>S-12000</td><td>Flourescent dye for protein staining, when detecting with SYBR Green II present, use excitation wavelength of 457 nm and emission wavelength of 670 nm</td></tr><tr><td>Copper grids</td><td>Electron Microscopy Sciences</td><td>G400-CP</td><td></td></tr></tbody></table>

purification of native complexes for structural study using a tandem affinity tag method

亲和纯化的方法已经成功地分离出原生复合蛋白质组学表征。结构的异质性和一定程度的复杂的成分异质性通常不妨碍进行这种研究的进展。与此相反，用于结构表征的复合应是既在组成和结构上均匀的，以及在比所需的蛋白质组学更高浓度的状态下进行纯化。最近，一直在电子显微镜的应用的大型大分子复合物结构的决心显著的进步。这加剧了利益的方法通过电子显微镜净化足够数量和质量结构测定天然复合物。串联亲和纯化（TAP）的方法进行了优化，提取和纯化的18亚基，从〜芽殖酵母0.8丙二醛核蛋白组件（ 酿酒酵母） </EM&gt;适用于负染色和电子显微镜低温。这里有详细的TAP方法，做出这些改变的理由所作的修改，以及采取的方法测定的在组成和结构均匀复杂。

使用了改良的方法TAP从S.净化酵母 U1的核蛋白，18亚基核糖核蛋白复合体。公布的协议2,3以下复杂的初始的TAP纯化，得到一个复合物，出现异类，迁移作为银三个频带染色天然聚丙烯酰胺凝胶（ 图2A）。在TAP方法的优化的多轮，产生了迁移，主要是一个单波段上指示更均匀组件（ 图2A）的天然凝胶的配合物。用荧光染料染色为...

Watch this Scientific Journal Video about Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method at JoVE.com

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

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 ...

purification-native-complexes-for-structural-study-using-tandem

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.5K 次观看.  Brandeis University. 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. 

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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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

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

从真核细胞蛋白质复合体串联亲和纯化

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

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

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 ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

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

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

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

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

<meta charset="utf8"></head><body>优化 FPLC 以高效纯化 His 标记的 FEN1 蛋白

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

<meta charset="utf8"></head><body>作者聚焦:优化 His 标记的 FEN1 蛋白的亲和层析

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化 

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

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 ...

生物学

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 ...

本机配合物的净化对结构研究使用串联亲和标签法

摘要

探索更多视频

此视频中的章节

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

从真核细胞蛋白质复合体串联亲和纯化

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

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

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

蛋白复合物的鉴定