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>

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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. 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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两个优点用于利用用于纯化天然复合物的TAP方法。但是，原始的TAP方法协议不设计成确保复杂的纯化是在组成和结构上是均匀的。因此在TAP方法已被修改，如上文详细描述的，为实现这一目标。 各个在TAP方法步骤进行了评估，以确定最佳的方法和条件，净化的组成和结构的均匀复杂。一个早期的关键步骤是细胞裂解。此步骤需要较高的收率，从而最大裂解人的愿望之间的平衡，同时确保所获得的复合物是高品质的， 即 ，是均匀的。不同的方法来裂解酵母细胞进行了探讨，包括使用珠打浆机，高剪切流体处理器，和球磨机。更便宜的咖啡研磨机的使用比其他方法的效率较低（40 - 60％的裂解），但复杂的分离和纯化出现单分散而几个这些方法似乎任扰乱颗粒完整性或造成一定程度的聚集。在结束时，未裂解的细胞的显著数目，以便确保得到高质量复杂。虽然40 - 60％的溶解是理想的酵母U1核蛋白，优化应用该协议，当一个建议<html>新的生物复杂。 这是至关重要的，以确定在该方法的步骤，可以加快纯化，以确保复杂的完整性没有被细胞蛋白酶，亚基解离损害作为稀释的结果，和TEV蛋白酶切割位期间在18℃延长温育。一个简单的步骤来实现时间高效净化是确保血脂并没有从早期的裂解液澄清结转，因为脂类重力纯化过程中阻止流动。其它步骤包括识别所述量树脂和列大小/尺寸的最佳平衡，从而达到有效的纯化和最大流率。一个特别重要的步骤是通过TEV蛋白酶，以减少对塔裂解时间。使用TEV蛋白酶在房子纯化，以确保自由和以高浓度17是蛋白酶/核酸酶。 TEV蛋白酶显著量使用，以获得在一个小时内完全切割。 内容】"&gt;在初始的TAP纯化的蛋白水解进行了观察。纯化期间蛋白质水解可以通过有效地工作，加入抑制剂的一个电池，并且包括在纯化高盐洗涤来限定。宽范围的在纯化的前半部分提供蛋白酶抑制剂的极大地改善了TAP的稳定标记的蛋白质和复杂的。此外，蛋白水解物通过细胞裂解和纯化的IgG的步骤中增加的300至500mM的单价浓度最小化。即使有上述修改到TAP方法，逐次洗脱级分断在其组成均一性的程度而变化的CBP亲和树脂的，因此，从第二，CBP层析步骤级分的选择性池，是必要的。 收到特定焦点的TAP方法的一个方面是所述非离子型洗涤剂NP-40的复杂的纯化，并在其高于CMC的浓度的重要性。 NP-4的存在下0在0.08％或更高的浓度对复合物的完整性和最终产量的总体有益效果。 NP-40的表观有益效果（多个）可能部分是由于减少树脂和复数之间的非特异性相互作用。而NP-40显示在纯化期间具有有益的效果，它可以被除去，纯化后，而不会导致复杂的聚集。 最后，为了优化结构研究在TAP方法关键是用于评估复合物的完整性和均匀性几个补充实验。这些措施包括光散射，SDS和本地PAGE使用TAP抗体Western blot和/或荧光染料染色，以及负染电子显微镜探测。本文详细介绍了TAP方法所做的更改，以产生足够数量的电子显微镜均匀复杂。额外的变化，可能需要对其他配合技术咨询方法进行，特别是阻止的NP-40的重要性mination。 综上所述，TAP纯化方法可以用来从合适的质量结构研究真核生物源成功净化大分子复合物。我们已建立了S的纯化的合适方法酵母 U1核蛋白和细节拍摄，以及理许多这些变化的步骤。对于其他复合物，我们建议研究者同样探讨在协议，其可以产生足够量的和复杂的质量的步骤。应检查的步骤包括细胞裂解，盐和添加剂的类型和浓度，包括洗涤剂，以及复杂的钙和金属螯合剂的EGTA灵敏度。此外，它是该复合物可以冷冻和解冻不扰动结构的长期研究的关键。重要的是，应该有几种可供选择的方式来测定所述复合物的结构中和之后纯化。一个本地人第二SDS-PAGE，负染色EM，和光散射我们如何最好地使用TAP方法纯化均匀复杂的调查都行之有效。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#916;1, leu2&#916;0, met15&#916;0, ura3&#916;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&#160;</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&#160;</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 &#956;L for a silver stained gel and 5 &#956;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&#160;</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&#160;</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。用荧光染料染色为蛋白质和核酸，它建立了纯化的复杂既有生物聚合物存在（ 图2B）。 纯化的复合物还通过SDS-PAGE和使用TAP标记抗体（ 图3A）Western印迹进行分析。与那提一致已经凝胶结果，根据已发布的协议纯化复杂表现出的TAP的蛋白水解标签蛋白（Snu71）。蛋白水解的量，然而，显著与TAP方法的修饰减少。几乎所有在U1的核蛋白复合十七蛋白质通过SDS PAGE分离并通过MALDI质谱法（ 图3B）清楚识别。以及天然和SDS-PAGE，复杂的质量在通过负染色电子显微镜（ 图 4）的纯化的不同阶段进行评估。从4C 图4A和4B观察到，但缺席的单分散颗粒是好样的结构研究的例子。这种分析方法提供了关键的见解，以改变在TAP方法的影响。 在TAP方法要求包含非离子型洗涤剂NP-40中，并在一个浓entration高于其临界胶束浓度（CMC）的。的评估在协议中描述的复合物的质量的方法的稳健性是公由其中两性离子表面活性剂的CHAPS，甘油或无共溶剂被取代的NP-40在所有的缓冲区（ 图5）的实验证实。母语PAGE和负染EM，U1的核蛋白出现与CHAPS降低稳定性最均匀和稳定的NP-40甘油不添加助溶剂。它可能是酵母U1核蛋白具有疏水表面和NP-40的防止聚合的通过涂覆该表面的存在。不幸的是，NP-40不能被透析除去，然后浓缩复杂将集中的NP-40。而较高的NP-40并未以扰乱复杂，但它并负颗粒的冻结在冷冻电镜和大胶束样聚集体玻璃体冰观察影响。洗涤剂吸收珠成功使用于remove没有出现在结构影响复杂广大的NP-40从U1核蛋白的样品。 以下，在该协议中，复杂的同时适合负染色和冷冻电镜详述的修饰的复合物的纯化，得到由粒子和独特的代表类的平均值（ 图6）的草坪的图像所证实。 <img alt="图1" src="/files/ftp_upload/54389/54389fig1.jpg" /> 图1.串联亲和纯化（TAP）方法概述。 （A）的两步法TAP示意图。感兴趣的（灰色）配合物包括一TAP标记亚基（黑色; U1核蛋白亚基Snu71）用由钙调蛋白结合肽的序列的基因组融合（CBP，绿色），一个识别位点的位点特异性蛋白酶的TEV（蓝色），和两个IgG结合蛋白A区域（PR值的<html>红）。在第一步骤中，细胞裂解物温育的IgG的树脂和复数经由其与蛋白A序列的相互作用保留。复从由TEV介导的裂解的树脂释放。在第二步骤中，将复合物温育以钙调素树脂， 钙依赖的反应。复杂的是通过添加钙离子螯合剂EGTA的释放。（B）继TAP印迹通过（A）所示的纯化步骤标签蛋白。样品被采取细胞裂解物中，第1步骤之后，并在TAP方法的第2步骤之后。在细胞裂解液中观察到的反应蛋白迁移第1步后速度更快，以下TEV蛋白酶裂解，因而蛋白质的序列的损失。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54389/54389fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> URE 2"SRC ="/文件/ ftp_upload / 54389 / 54389fig2.jpg"/&gt; 图2. TAP方法的优化。 （A）银染TAP纯化复合物的天然凝胶。乡土PAGE:蛋白质标准（泳道1）;复杂的按原来的TAP方法3（泳道2）进行纯化，三个频带观察到（箭头）;从相同的TAP净化以下改变缓冲三连冠洗脱，两个波段观测（箭头）（泳道3 - 5）;进一步的修改在TAP方法，主要得到一个单一的复合带在约800 kDa的迁移（泳道6）。（B）的纯化的复合物的组合物分析染色的单一凝胶泳道与蛋白质和RNA的反应性污渍。的变性PAGE:用蛋白质荧光染料染色的蛋白质标准（泳道1）;复合物与蛋白荧光染色（泳道2），观察到两个条带成像;在泳道2相同车道，现在成像用，两个频带观察到核酸荧光染色（泳道3）;和荧光信号在躺在2,3泳道（泳道4）染色情结。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54389/54389fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54389/54389fig3.jpg" /> 图3. TAP方法的优化。 （一）在复杂的质量改进Western blot分析，以下TAP标记亚单位Snu71探测与α-TAP抗体。几个蛋白水解频带是初始可见（泳道1），但是改进减少低频段的数量相当（泳道2和3）。（B）代表的SDS凝胶用荧光蛋白凝胶染料染色。凝胶解决复杂的17蛋白质亚基12。在乐队中的蛋白质的鉴定通过质谱确定。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54389/54389fig3large.jpg" target="_blank">请点击此处查看LARG</a>呃版本这个数字。 <img alt="图4" src="/files/ftp_upload/54389/54389fig4.jpg" /> C（（A）TAP的第 1步;（B）TAP的2步: 在该TAP方法中的阶段图4.样品的质量负染色电子显微镜通过复杂的后负染电子显微镜评估的范围。 ）复杂的不稳定。比例尺，125纳米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54389/54389fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54389/54389fig5.jpg" /> 图5.影响对添加剂在TAP中的方法，缓冲器现状的样本质量泳道1 - 4的c的TAP纯化的最后或第 2步的前四个洗脱在存在omplex纯化:（A）中的非离子型洗涤剂壬phenoxypolyethoxylethanol（NP-40），通过非变性PAGE（顶部）和负染色的EM（底部）分析;（B）中的两性离子洗涤剂3 - [（3-胆酰胺丙基）二甲氨基] -1-丙烷（CHAPS），通过非变性PAGE（顶部）和负染色的EM（底部）分析;（C）甘油，通过非变性PAGE（顶部）和负染色的EM（底部）分析;和（D）无添加剂，通过非变性PAGE（顶部）和负染色的EM（底部）进行分析。比例尺，50纳米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54389/54389fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54389/54389fig6.jpg" /> 图6. TAP方法纯化可视化复杂。 （A）顶部，负染色电子microsco的形象代表PY（EM）显微照片。观察到的是类似形状颗粒的草坪，两个是盒装，通过优化的TAP方法纯化。底部，选择采取突出粒子特色粒子的平均班的。比例尺，50纳米。（B）顶部，一个冷冻电镜显微照片的形象代表。观察到的具有代表性的颗粒更高的对比度的区域，看到盒装两个这样的区域。玻璃体冰封选择颗粒的底部，班级的平均。比例尺，50纳米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54389/54389fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

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

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

A型流感病毒的核蛋白复合物的分离纯化及可视化

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

科研

生物学

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

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 ...

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 ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-他的净化:一个两步亲和纯化协议屈服全长纯化的蛋白质

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...