Name: protein complex affinity capture from cryomilled mammalian cells
Uploaded: 2016-12-09T13:00:00.000+00:00
Duration: 10 min 37 s
Description: Watch this Scientific Journal Video about Protein Complex Affinity Capture from Cryomilled Mammalian Cells at JoVE.com

我们感谢布赖恩T.蔡特教授的宝贵意见，支持和获得MS仪器，以及R.凯利女士莫洛伊优秀的技术支持。我们感谢凯利裸女士与副本编辑支持。这项工作是由美国国立卫生研究院资助P41GM109824，P41GM103314和P50GM107632的部分资助。

1.细胞收集和冷冻<ol><li>使用适当的培养条件为感兴趣25,26的细胞系生长1-8 G细胞的材料。这个协议是最多8克细胞（〜10 9个细胞），从参考文献19,27,28改性优化。通常，〜5克的HEK-293或HeLa细胞可以从生长8500厘米2培养板，以〜90％汇合得到的。 注意:这些协议使用液氮（LN 2），能引起严重的低温灼伤的。唐防护服和运动适当的处理措施。 </li><li>倒出生长培养基（废物）成一个大烧杯中。 </li><li>广场上冰的培养皿中的一个大的矩形冰锅。 </li><li> 20毫升冰冷的1X磷酸盐缓冲盐水（PBS）添加到培养皿，并用大细胞刮刀释放从盘的细胞;细胞转移到50毫升管中，预先在冰上冷却;举办冰上管。 注意:对于所有细胞操作步骤使用电动移液器设置为"低"和25ml移液器，以避免在传输操纵细胞过度剪切。安排50毫升收集管，并在冰桶启动过程之前1倍PBS中。 </li><li>另外的10毫升冰冷的1×PBS中的添加到同一个盘。收集剩余的细胞，并将它们转移到50毫升管。 </li><li>重复每道菜;从不同的菜细胞悬液可被组合，以减少取样数和废塑料。 注意:由于细胞本身不会构成悬架体积的相当大的比例，三个板值得细胞悬浮液的可组合成两个50毫升管。因为50ml试管实际持有比标称容量多，八板通常可以在五个这样的管蔓延。 </li><li>离心机在1000 xg离心，4℃5分钟。 </li><li>小心倒出上清。重悬在10毫升冰冷的1×PBS中每个粒料。巩固地区环境部门一道spended粒料，最多5每50毫升管，以减少取样数。 </li><li>离心5分钟在1000 XG，4℃。 </li><li>小心倒出上清。重悬在10毫升冰冷的1×PBS中的沉淀</li><li>从20毫升注射器中取出柱塞并将它放在一边。帽注射器的喷嘴和细胞悬浮液转移到它。 </li><li>放置注射器50毫升管中并离心5分钟内以1000 xg离心，4℃。 </li><li>吸出上清液与连接到一个真空阱系统，直到刚刚细胞的顶层细尖吸管开始被吸上来。这导致在潮湿的细胞沉淀。 </li><li>开盖注射器，插入柱塞并直接滴落细胞进入的大塑料烧杯装满LN 2，在一个聚苯乙烯泡沫塑料箱的LN 2浴保持。强制沉浸从注射器的剩余细胞。 </li><li>通过倾倒转移冷冻的细胞至50ml管中。松散帽管，以让多余的液氮蒸发;担任船长在-80℃下摆过夜。完全拧紧瓶盖的第二天。将冷冻的细胞可在-80℃保存以这种方式，直到低温球磨。 注意:所有的LN 2已明显蒸发之前，不要盖紧管，否则过大的压力可能会导致管爆炸。不封闭管中的液氮消散后，可能会导致对细胞材料霜的积聚，加入过量的水的重量，有效地减少了在碾磨的蛋白浓度。 </li></ol> 2.冷冻细胞的破坏低温注:铣削和操作所有工具应预先冷却，2 LN。一个小酒瓶将需要添加的jar在2 LN和浇2 LN在封闭的研磨罐的顶部。自制工具显示在参考19。低温手套将需要处理LN 2冷却研磨设备。铣削这里使用的罐子的盖子（见材料表 ）通常安装有橡胶垫船;这将需要使用市售的聚四氟乙烯盖密封垫片可靠地执行以下方案来代替。此外，由于由气态N 2压力可在研磨过程中建立起来的罐子内的较高水平，我们建议您安装市售的5条/ 500千帕的压力阀作为安全释放预防措施28。 <ol><li>从-80℃贮存去除细胞珠和持有50毫升管架在液氮浴。 </li><li>预冷却50毫升的容器，二20毫米球，和盖在含有2 LN干净矩形冰桶。如果使用一个预冷却所述聚四氟乙烯（PTFE）绝缘体;任一组的冰球（见附图27），或一个套筒和-冰球（ 见图2）。预冷却完成时LN 2镇静的剧烈沸腾。 </li><li>上设置适当的平衡磨。 注意:这将等于罐子，瓶子盖的质量，所使用的研磨球，和要加入到该罐的细胞的数量。如果使用任何PTFE绝缘体，它们的质量也应包含在内。我们建议记录瓶，盖的群众，和球（和它们的组合质量，包括使用的任何绝缘体）提前和记录它们存储磨坊附近一个信息表。 </li><li>使用镊子，将两个预冷却20毫米研磨球和冷冻细胞预冷研磨罐内。 注:由于罐子，球表面研磨过程中材料的小损失，回收的材料会随着细胞的研磨的质量增加的百分比。所描述的方法，材料损失是非常温和的，是-0.3克湿重细胞（WCW）的顺序。制造商的指导方针指示样品的最大体积装载应罐体积的〜1/3 29球的总体积应为〜1/3和Remaining〜1/3个免费空间是用于球的自由流动。 </li><li>加入2 LN的罐子高达约半满，放在罐子盖和装配转移到磨。 注意:如果使用一个首先安装选定的绝缘体。 </li><li>钳装配到位，并执行使用下面的程序铣削三个周期:400转，3分钟，反向旋转，每分钟，无间隔休息。冷静了2 LN的研磨罐在每个周期之间。 注:在铣削球在行星运动罐子内碰撞产生不同的clunking噪音。如果没有听到这些声音，铣没有发生。终止铣削循环，不要指望这个周期，将罐子装配回2 LN并检查罐子的内容。确保没有冰已经形成，如果它有，用预冷却的抹刀芯片其从罐壁离开。从步骤2.5再次启动。 如果不使用绝缘体或绝缘体的冰球，将罐子装配回LN <sUB&gt; 2周期之间重新冷静，加2至LN〜½满各一次。该LN 2加入将罐子作为罐子增加铣削时的温度范围内蒸发。这导致了罐中的压力积聚。因此，脱离自磨机罐子时，松开钳非常缓慢。如果夹紧迅速释放，细胞粉可以与快速减压逸出。夹具的缓慢释放允许从在温和的，受控的方式将罐逸出的压力，并防止细胞材料的损失。气态N 2柔和的逃生经常可以听到嘶嘶的钳慢慢释放-这是正常的。 如果使用的套筒和冰球绝缘体，罐子组件可以在磨机左和LN 2加入到绝缘体/罐组件在原地 。 </li><li>移动罐回到2 LN，并让暂时休息降温。如果使用的套筒和冰球绝缘体，罐子可以从内套去除第i个2铲的帮助下，对任何一方提供的杠杆作用。一旦罐被LN 2休息，小心取出盖子，使用镊子除去滚珠，并用预冷的刮刀将粉末转移到预冷的50ml管中。加入少许LN 2的开瓶/粉可以帮助移除该结块到粉球，删除它们之前。 注意:一旦罐已经打开，取出球前2 LN少量添加到它。这将有助于恢复结块的表面上的粉末。细胞粉末应在-80℃或更低直至使用举行。在我们的经验，细胞粉可以存储在这种方式，基本上无限期，在不影响性能。 </li></ol>从细胞提取物蛋白复合物3.亲和捕获注意:以下协议实现了与感兴趣，共同的蛋白相互作用包括亲和配体的亲和介质，诱饵，或抗体upled至微米级的顺磁珠。这些可以在内部19,30来制备，使用商购的试剂盒，或购买作为现成的试剂。 <ol><li>细胞提取物的制备 注意:当处理细胞粉，记得用器皿和管预冷与2 LN。含电池粉管时，应该始终不能在-80℃举行2 LN。放置含细胞粉末在管架，在一个泡沫聚苯乙烯容器LN 2 50毫升管（多个）。 <ol><li>称出100毫克的细胞粉的成1.5或2 ml离心管。 注:我们观察到细胞的这个量是一个很好的起点，通常会得到目标蛋白在几十到几百毫微克以每秒数千份亲和捕获的适度表达的蛋白（〜50kDa的质量后的范围，目前细胞），假定目标的提取和捕获效率是≳70％。这样的收益率提供了直接visualiz通过SDS-PAGE和蛋白染色的纯化的级分的通货膨胀。 </li><li>去皮分析天平与空离心管。细胞分装成粉用液氮冷却匙或锅铲管。检查分析天平管内分配的粉末的质量。 注意:为了方便称出细胞的粉末，也可以使用小体积测量匙。这些已发现，得到可重复的结果（见附图19）。我们发现用螺丝帽或&#39;安全锁&#39;离心管中最好的结果。压力从蒸发LN 2可能进入管会导致标准离心管，只是在加入萃取溶液之前弹出后续变暖期间开-潜在地导致样品的损失。 </li><li>打开管（或拧松螺丝帽）与细胞粉末静置在室温1分钟。这样就释放了管内的压力，并防止抽出的立即冻结溶液时添加到该粉末中。这1分钟孵化过程中没有解冻观察。 </li><li>加入400微升萃取辅以蛋白酶抑制剂和漩涡简单地说，然后在冰上举行，同时继续执行步骤3.1.5。样品应全部随后的操作之间举行冰上直到洗脱。 注意:提取剂的最佳组合物取决于蛋白质复合物（ES）来进行纯化。一些一般性的指导表1和支持提供参考。 </li><li>使用微尖超声发生器给样品一个简短的低能量脉冲驱散任何集合体。 （超）超声有4个脉冲（每2秒，2 A;约15-20总能量J） 注:涡旋分送小区粉末进入萃取剂，但根据不同的解决方案，字符，一些聚集体可以观察到。我们已发现，分散这些聚集体提供了随后的亲和捕获期间的最佳产率。一个简短的微尖SONI阳离子分散轻易这些聚集体。该解决方案应该出现半透明，但均匀，无明显聚集。水浴sonicators往往过于低功率高效地分散这种聚集而不显著更长的样品处理时间，但可以是适于与适当的设置。 </li><li>通过离心澄清提取物（ 如20,000 xg离心，10分钟，4℃）。 注意:澄清的细胞提取物的等分试样可被保存用于比较到颗粒中，亲和捕获后上清液中的内容，并从亲和介质洗脱后获得的级分，以确定提取的目标蛋白质的效率和捕获， 例如，通过免疫印迹（见讨论）。 </li><li>去除上清（澄清的提取物），并继续进行亲和捕获。 注:在SDS-PAGE上样缓冲液将沉淀可以重新萃取在70℃，以确定目标蛋白的D释放的程度通过比较在步骤3.1.6中获得的提取物uring初始非变性提取。 </li></ol></li><li> 亲和捕获 <ol><li>预洗磁亲和力网上平台。这可以在细胞提取物被离心来完成。 </li><li>放置在磁体的管（多个）。珠粒会积聚在管的秒内的侧面，允许要删除的存储解决方案。 </li><li>加入500μl的提取液来珠。简言之涡结构中速（足以悬浮）。脉冲旋管在微型离心机收集所有内容的底部。这样做可以确保解决方案的最低结转。放置在磁体管和吸出溶液。 注:具有鲜明特色的磁介质可从各种商业供应商。结果可以根据这些特征，包括珠尺寸，均匀性，表面涂层，和抗体偶联化学变化。并排-SI在应用程序中去解决审判上的选择试剂之前推荐的。 </li><li>经澄清的细胞提取物转移到含有-预洗的亲和力中，涡旋简单地悬浮1.5-2.0毫升管发起亲和捕获。 </li><li>孵育在4℃下与一旋转轮连续温和混合30分钟;珠应在整个孵化继续停牌。 </li><li>脉冲旋管在微型离心机收集所有内容到管的底部。吸出上清液，并在步骤3.2.3描述用1ml冷提取液洗珠三次。放置在管上的磁铁。除去溶液。继续添加下一个解决方案，并重复。在第2 次洗涤，通过移液转移珠和洗涤溶液一起到新鲜的离心管。这降低样品污染，在洗脱的点，通过用于与叔孵育吸附在管的壁无规蛋白他细胞提取物。第3 次洗涤后，将珠粒应脉冲纺丝简要地在微型离心机收集所有内容的底部。将管回到了磁铁，并删除任何残留液体。这使得洗脱，确保洗脱的样品将有统一的卷之前去除溶液的最后几微升。它也保证了洗脱将是有效的，作为洗脱溶液将不会被剩余洗涤稀释;这种残基也可以向盐效果和改变蛋白质在SDS-PAGE迁移（见下文）。 注:后结合物上清液的等分试样可被保存用于比较在3.1.6中产生的澄清的提取物。通过免疫印迹。结果将表示从细胞提取物中的靶蛋白的耗尽的程度。 </li><li>洗脱在任何一个天然或变性的方式;考虑战略最好的下游应用1。 注:本地洗脱strategie的细节旨意取决于所用的亲和标签（见讨论）。对于大多数用户来说，变性，用SDS-PAGE样品缓冲液随后将样品通过SDS-PAGE用蛋白染色31的分析洗脱，将是最实用的初始方法。 注:样品缓冲液将取决于电泳系统上的组合物被使用。许多实验室投下自己的Tris甘氨酸凝胶，利用电泳断续和拉姆力缓冲系统32-34。一个常见的样品缓冲液配方（1×），包括:10％（重量/体积）蔗糖，50毫摩尔DTT，2％（重量/体积）SDS，62.5毫摩尔Tris-氯pH值8.8，0.0004％（重量/体积）溴酚蓝31 。我们建议准备1.1倍的股票，省略DTT。 1/10的500mM DTT的体积应的SDS-PAGE之前，加入到样品中（见下文）。许多市售的SDS-PAGE系统也可用;这些可以包括特定于系统的样品缓冲液。 </li><li>添加SDS-PAGE样品缓冲液中不还原剂（以减轻释放从珠粒抗体链）和孵育在室温下搅拌5-10分钟。 注意:这将与大多数基于亲和力的相互作用干扰，释放捕获的蛋白质到上清液。更持久的相互作用可能从高温受益释放（通常70℃就足够了）。 </li><li>收集并保存上清。样品可以在-20℃下短暂存储（几天）被冻结或-80℃下长期储存，直到分析是需要的。 </li><li>若使试样进行SDS-PAGE，接着用标准技术31蛋白染色。 MS可以用于表征样品组合物1。个别蛋白质条带可以被切除以识别或整个样本可通过电泳样品只简单（4-6毫米到凝胶），并与样品中一起处理所有蛋白质作为来表征在一个单一的分析"凝胶塞。" 注:初始前加入DTT婷电泳。如果计划进行MS，样品可以是电泳后35烷基化;然而，这是经常电泳36之前更方便烷基化物。 </li></ol></li></ol>

The authors declare that they have no competing financial interests.

这三个协议协力工作以（1）通过低温球磨制备固态破损的细胞，（2）实现在行星式球磨机破损，以及（3）产生提取从细胞粉末与亲和捕获目的蛋白质在复合其生理作用因子。许多不同的细胞裂解技术存在，包括采用冲击粉碎，剪切和/或压力机械/物理方法，以及化学/酶学方法，各有不同的优点和缺点49,50。每个调查员鼓励探索最适合自己的方法分析，牢记细胞破损和蛋白提取任意选择的方法很可能会推出偏见51,52迫使经验优化（见下文）。机械方法可以产生高热量和/或剪切力可破坏蛋白质复合物。通过低温球磨采用凭借在t样品的液氮冷却避免了热效应该方法的他持续时间。行星球磨机被理解为依靠影响力和摩擦力，包括剪切应力，随着粒径的减小53,54的组成部分。在设置报道我们没有观察到蛋白质复合物变性。事实上，我们已经提取并回收显然完好〜50丙二醛核孔复合物11和酶不是采用&#39;温柔&#39;去污剂裂解7筹备参展更高的比活性的活性反转录转座子。细胞裂解的化学品/酶学方法从限制，即该单元的内容被释放到支持细胞膜和结构的大分子的破坏，但可能不适合于维持所述蛋白质复合物的完整性的体外环境（ES遭受） 出于兴趣。频繁，既不形成与感兴趣的蛋白质复合物的成分，也没有必要的条件，以稳定在目标配合物是预先已知的。固态铣的一个主要好处是，破碎和提取解耦，允许源材料中不含添加液体制备，贮存（-80℃以下），聚敛，方便按需检索实验; 例如，为了在亲和捕获体外条件探索优化。蛋白质的相互作用是在高浓度55,56最稳定的，因此最小化萃取剂的体积可以用于保持生理相互作用是有利的。另一方面，也有实际的考虑 - 蛋白质复合物就需要分区出细胞和成非粘性的水相，无不溶聚集体，以混在一起的亲和力介质靶复合物。此外，需要一种用于系统化和重复性在体外环境（pH值，盐浓度等 ）一些标准化和控制。我们发现，提取公关oduced中的稀释范围1:4-1:9（W:V）满足实际和理论问题，产生高质量的结果。另外，也可以测定细胞提取物的最佳比例，以亲合介质的需求。这是根据经验通过提取物的滴定用不同量的亲和力介质的完成，可对试验的信噪比检测的效果，下面进一步讨论。靶蛋白的一个极好的消耗通常是靶蛋白的可溶部分的≥70％，但&gt; 90％是可取的，可以是与提取条件小心参数实现的。许多这样的实际考虑覆盖在参考文献1。顺磁性珠粒在一个专门的微离心管架使用钕磁铁操纵，虽然自制替代方案是可行的。当放置在支架内时，小珠收集在管的磁场的影响下的一侧。溶液然后可未经二除去sturbing珠。 所提出的低温球磨协议的一个限制，以此处使用的行星式球磨机显影（见材料数据表 ），是材料的最小量以有效轧机和使用该装置（&gt; 1克）回收细胞的粉末。这样的量很容易与许多微生物，细胞系，和模式生物获得，并且也经常与从实验室动物切下的组织来实现。然而，某些细胞系可能是非常困难的丰度和动物组织生长可能是稀少。材料可以将少量使用利用较小的容器的其他设备所取得的粉末细度的牺牲可比研磨，虽然可能。此外，机械研磨设备的成本可以是用于某些实验室昂贵。使用有害低温球磨可以使用多种替代设置14-19来实现，其中最经济地用手乐和迫击炮，虽然破损效率大幅下降。亲和捕获的协议典型地瞄准细胞裂解的具有高效率，以促进最大蛋白质提取到溶液中，因此，为对靶蛋白质复合物的捕获最大的潜力。另一方面，已经证明在体外剪接酵母提取该最大裂解可以不与体外生化活性57,58最大等同。我们并没有在我们迄今已经测试了系统观察到这样的问题，因此，在低温球磨不刻意限制我们的细胞破裂。然而，这种可能性应该考虑优化体外酶测定时承担。虽然低温球磨为破碎细胞的高效方法，超声处理有限数量往往有利于从哺乳动物组织生产均匀全细胞提取物，因为不均匀的聚集物有时可以通过视觉检查来观察:典型地在萃取条件下，使用低到中等盐（100-300毫米）和非离子型洗涤剂（0.1-1％V / V）的浓度。因为我们观察到，这些聚集体的存在可减少随后的亲和捕获的产率和/或质量，我们经常实施超声处理以分散它们（即使当它们不是用肉眼观察到的）。聚集体与团聚膜碎片，可比以前从研磨酵母细胞中提取58报道的一致。超声处理也用于某些协议剪切DNA和解放染色质片段成溶液，然而，在该协议施加超声处理的程度没有明显的片段的DNA。优异的亲和配体或抗体针对感兴趣的特定蛋白质的有限可用性（或成本高）可以是另一种障碍。市售的亲和试剂的广泛可利用，当模式生物是适合于基因组标记或TRansfection异位表达载体，允许的利益，因为亲和标记的融合蛋白质的表达。但是，生产定制的抗体已经越来越可行和多克隆和单克隆抗体可以在亲和捕获应用优异的性能。这些多方面的考虑，也包括在更详细的参考1。 如何亲和力介质的细胞裂解的方法和选择会影响结果的例子在图1中示出。由不同的萃取剂施加影响的例子可在参考文献1,11,24中可以看出。因为这些和其它实验参数影响亲和捕获的质量，使得难以辨别的FP，"珠蛋白质组"和计算方法的库，以消除非特异性的污染物已被开发，以协助确定的FP 40-43。然而，这种方法只substit尤特最佳样品制备在有限的程度59。观察最佳实践和经验优化亲和捕获实验将提供最优质的样本进行下游分析，下面进一步讨论。可提高样品纯度容易实现的做法是原生洗脱。天然洗脱最常用于获得亲和力分离的蛋白质复合物，完整的，用于进一步的实验;但是，因为它经常提高样品的纯度，但也可以用于单独这个原因。然而，如在图1中 ，面板II表明，天然洗脱来改善样品纯度的能力可取决于亲和力培养基在细胞提取物中的目标蛋白质的丰度的量的精确滴定-当介质是在过量，未被占用的互补位可以允许FP蛋白在原生洗脱级分观察到的脱靶的积累。使用此处描述的试剂和方法，它ħ如我们的经验是FP的我们的实验的一个最大的贡献者是被标记的蛋白质本身一旦从活细胞的上下文中除去。 （图1.II与图S1）。在这样的情况下，即在不存在抗原的产率无关蛋白质组亲代细胞系的控制是没有实际价值的;同样，对于标签的唯一或穗在那些缺乏什么，但靶抗原的控制。因此，每当实际我们实现的I-DIRT 7.38，直接测量用定量质谱裂解后-蛋白质相互作用的积累。在协议中的步骤3.2.7所提到的，用于本机洗脱程序将与所使用的亲和系统的细节而变化。天然洗脱最常由标签的标记的蛋白质复合物或蛋白水解切割的有竞争力的位移来实现，释放从亲和介质的复合物。由小表位标签的亲和力几个系统的存在，FOR，它的表位本身可作为肽用于标记蛋白质复合物60的竞争性洗脱有用。同样地，几种蛋白酶可特异性裂解的同源位点策略性地放置在亲和力标记的融合蛋白61。取决于所选择的亲和系统的具体情况，适当的洗脱方案可以被采用。 通常，所得到的结果的质量会显著由样品制剂的质量的影响。重要的是要通过这些协议的每一步仔细和精确地工作，并尽可能快速地，同时维持护理和精度。最好是通过每个步骤来跟踪感兴趣的蛋白质的分区来了解每个操作的效率。例如:如何丰富是在所讨论的细胞或组织目的蛋白质？也许正在研究（及其复合物）的蛋白质将是具有挑战性的质量来表征法因低丰度。如果纯化的复合物是由一般的蛋白染色（约纳克范围）检测，质谱法是有可能成功。如果感兴趣的捕获蛋白质只能由敏感增强化学发光Western印迹（约皮克范围）进行检测，质谱是不太可能是有效的。即使感兴趣的蛋白质是在细胞内丰富，如何丰富是它在细胞提取物产生的？是否蛋白分区的溶液或者是它在沉淀中？如果是后者，一个新的提取溶液可设计可提高回收率。一旦进入细胞提取物与亲和介质结合，如何有效地被该蛋白质捕获？是否仍然蛋白通过以后的洗涤约束？那么其他copurifying蛋白质？通过保存在协议的适当步骤的每个样品的等分试样这些问题可以容易地回答，一般是通过Western印迹针对感兴趣的蛋白或一般的蛋白质染色，但其它测定法也可以是合适的。优化每一步骤将提高捕获复合物的产率和纯度，虽然有可能在最大化一个属性或其他要作出折衷。 亲和捕获的许多研究人员的一个典型应用是在一个小数目的感兴趣的蛋白质的体内相互作用物鉴定候选;这些候选人通常经受验证通过正交方法在体内表现出的物理交互件的生物学意义。亲和捕获也由高通量研究用于产生copurifying一个（几乎）蛋白质组范围的基础上观察到的蛋白质，有利于有关体内络合物推定计算推断列表。这些研究的许多例子可在文献中找到。这种方法foregoes的捕获条件的优化对于任何给定的目标有利于探索的人ÿ目标;因此，推断络合物本身很少检索到的任何给定样品中充分完整和高纯度。相反，在许多不同的亲和捕获的样本之间观察到组合物的部分重叠用作推论42的基础。这两种方法提供宝贵的数据，我们对相互作用组的认识。然而，亲和捕获的一个主要的好处是，它不会经常提供的机会，以获得完整的和高度纯化的，其前提是作出努力来优化该过程的复合物。我们认为，该方法的未来在于增加的容易性和优化的内源性蛋白复合物11的捕获效率，对于生理交互件和在进一步的下游生化，酶学更经常地使用，和结构研究的色域的更准确的评估， 如引用7,9,23。

A correction was made to: Protein Complex Affinity Capture from Cryomilled Mammalian Cells. The References section has been updated from:
<ol start="29">
	<li>Cristea, I. M., &#38; Chait, B. T. Conjugation of magnetic beads for immunopurification of protein complexes. Cold Spring Harbor Protocols. 2011 (5) (2011).</li>
	<li>Rosenberg, I. M. Electrophoretic Techniques. Protein Analysis and Purification. (4), 63&#8211;117 (2005).</li>
	<li>Ornstein, L. Disc Electrophoresis. I. Background and Theory. Ann N Y Acad Sci. 121 (A2), 321&#8211;349 (1964).</li>
	<li>Davis, B. J. Disc Electrophoresis. II. Method and Application to Human Serum Proteins. Ann N Y Acad Sci. 121, 404&#8211;427 (1964).</li>
	<li>Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227 (5259), 680&#8211;685 (1970).</li>
	<li>Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., &#38; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature protocols. 1 (6), 2856&#8211;2860 (2006).</li>
	<li>DeGrasse, J. A., Kalkum, M., Krutchinsky, A.N., Padovan, J. C., &#38; Zhang, W. MALDI Sample Preparation. rockefeller.edu at &#60;http://prowl.rockefeller.edu/protocols/in-gel-digestion.html&#62; (2006).</li>
	<li>Lubas, M., Christensen, M. S., et al. Interaction profiling identifies the human nuclear exosome targeting complex. Mol Cell. 43 (4), 624&#8211;637 (2011).</li>
	<li>Tackett, A. J., DeGrasse, J. A., Sekedat, M. D., Oeffinger, M., Rout, M. P., &#38; Chait, B. T. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J Proteome Res. 4 (5), 1752&#8211;1756 (2005).</li>
	<li>Wang, X., &#38; Huang, L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry. Mol Cell Proteomics. 7 (1), 46&#8211;57 (2008).</li>
	<li>Trinkle-Mulcahy, L., Boulon, S., et al. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 183 (2), 223&#8211;239 (2008).</li>
	<li>Boulon, S., Ahmad, Y., et al. Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics. 9 (5), 861&#8211;879 (2010).</li>
	<li>Armean, I. M., Lilley, K. S., &#38; Trotter, M. W. B. Popular computational methods to assess multiprotein complexes derived from label-free affinity purification and mass spectrometry (AP-MS) experiments. Mol Cell Proteomics. 12 (1), 1&#8211;13 (2013).</li>
	<li>Mellacheruvu, D., Wright, Z., et al. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods. (2013).</li>
	<li>Cheeseman, I. M., &#38; Desai, A. A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci. STKE 2005 (266), pl1 (2005).</li>
	<li>Goldberg, S. Mechanical/physical methods of cell disruption and tissue homogenization. Methods in Molecular Biology. 424 (Chapter 1), 3&#8211;22 (2008).</li>
	<li>Grabski, A. C. Advances in preparation of biological extracts for protein purification. Methods in Enzymology. 463 (C), 285&#8211;303 (2009).</li>
	<li>Dhabaria, A., Cifani, P., Reed, C., Steen, H., &#38; Kentsis, A. A High-Efficiency Cellular Extraction System for Biological Proteomics. J Proteome Res. 14 (8), 3403&#8211;3408 (2015).</li>
	<li>Glatter, T., Ahrn&#233;, E., &#38; Schmidt, A. Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells. J Proteome Res. 15 (2), 679 (2016).</li>
	<li>Zhao, Q.-Q., Yamada, S., &#38; Jimbo, G. The Mechanism and Grinding Limit of Planetary Ball Milling. KONA. 7, 29&#8211;36 (1989).</li>
	<li>Sheng-Yong, L., Qiong-Jing, M., Zheng, P., Xiao-Dong, L., &#38; Jian-Hua, Y. Simulation of ball motion and energy transfer in a planetary ball mill. Chinese Phys. B 21 (7), 078201 (2012).</li>
	<li>Fulton, A. B. How crowded is the cytoplasm? Cell. 30 (2), 345&#8211;347 (1982).</li>
	<li>Ellis, R. J. Macromolecular crowding: obvious but underappreciated. Trends Biochem Sci. 26 (10), 597&#8211;604 (2001).</li>
	<li>Kalkum, M. Using the Retsch MM301 Ball Mill for Cryogenic Disruption of Yeast Cells. 2 City of Hope, (2003).</li>
	<li>Staley, J. Making Whole Cell Extract of Saccharomyces cerevisiae cells using the Retsch MM301 Ball Mill. Retsch GmbH: (2005).</li>
	<li>Bell, A. W., Nilsson, T., Kearney, R. E., &#38; Bergeron, J. J. M. The protein microscope: Incorporating mass spectrometry into cell biology. Nat Methods. 4 (10), 783&#8211;784 (2007).</li>
	<li>Zhao, X., Li, G., &#38; Liang, S. Several affinity tags commonly used in chromatographic purification. J Anal Methods Chem. 2013 (1), 581093&#8211;8 (2013).</li>
	<li>Waugh, D. S. An overview of enzymatic reagents for the removal of affinity tags. 80 (2), 283&#8211;293 (2011).</li>
	<li>Williams, R. J., &#38; Lyman, C. M. A neutral buffered standard for hydrogen ion work and accurate titrations which can be prepared in one minute. J Am Chem Soc. 54 (5), 1911&#8211;1912 (1932).</li>
	<li>Johnson, M. Detergents: Triton X-100, Tween-20, and More. labome.com. 3 (163) (2013).</li>
	<li>Deppert, W. R., &#38; Luka&#265;in, R. Buffers and Additives. Journal of Chromatography Library. 61 (C), 839&#8211;862 (1999).</li>
	<li>Ugwu, S. O., &#38; Apte, S. P. The Effect of Buffers on Protein Conformational Stability. Pharmaceutical Technology. 28 (3), 86&#8211;108 (2004).</li>
	<li>Linke, D. Detergents: an overview. Methods in Enzymology. 463 (34), 603&#8211;617 (2009).</li>
	<li>Zhang, J. Protein-Protein Interactions in Salt Solutions. Protein-Protein Interactions Computational and Experimental Tools. (18), 359&#8211;376 (2012).</li>
</ol>
to:
<ol start="29">
	<li>Ball Mills - Guidelines for sample amount and ball charge. 1&#8211;3at &#60;http://www.retsch.com/products/milling/ball-mills/planetary-ball-mill-pm-100/information-downloads/&#62; Retsch GmbH (2014).</li>
	<li>Cristea, I. M., &#38; Chait, B. T. Conjugation of magnetic beads for immunopurification of protein complexes. Cold Spring Harbor Protocols. 2011 (5) (2011).</li>
	<li>Rosenberg, I. M. Electrophoretic Techniques. Protein Analysis and Purification. (4), 63&#8211;117 (2005).</li>
	<li>Ornstein, L. Disc Electrophoresis. I. Background and Theory. Ann N Y Acad Sci. 121 (A2), 321&#8211;349 (1964).</li>
	<li>Davis, B. J. Disc Electrophoresis. II. Method and Application to Human Serum Proteins. Ann N Y Acad Sci. 121, 404&#8211;427 (1964).</li>
	<li>Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227 (5259), 680&#8211;685 (1970).</li>
	<li>Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., &#38; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature protocols. 1 (6), 2856&#8211;2860 (2006).</li>
	<li>DeGrasse, J. A., Kalkum, M., Krutchinsky, A.N., Padovan, J. C., &#38; Zhang, W. MALDI Sample Preparation. rockefeller.edu at &#60;http://prowl.rockefeller.edu/protocols/in-gel-digestion.html&#62; (2006).</li>
	<li>Lubas, M., Christensen, M. S., et al. Interaction profiling identifies the human nuclear exosome targeting complex. Mol Cell. 43 (4), 624&#8211;637 (2011).</li>
	<li>Tackett, A. J., DeGrasse, J. A., Sekedat, M. D., Oeffinger, M., Rout, M. P., &#38; Chait, B. T. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J Proteome Res. 4 (5), 1752&#8211;1756 (2005).</li>
	<li>Wang, X., &#38; Huang, L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry. Mol Cell Proteomics. 7 (1), 46&#8211;57 (2008).</li>
	<li>Trinkle-Mulcahy, L., Boulon, S., et al. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 183 (2), 223&#8211;239 (2008).</li>
	<li>Boulon, S., Ahmad, Y., et al. Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics. 9 (5), 861&#8211;879 (2010).</li>
	<li>Armean, I. M., Lilley, K. S., &#38; Trotter, M. W. B. Popular computational methods to assess multiprotein complexes derived from label-free affinity purification and mass spectrometry (AP-MS) experiments. Mol Cell Proteomics. 12 (1), 1&#8211;13 (2013).</li>
	<li>Mellacheruvu, D., Wright, Z., et al. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods. (2013).</li>
	<li>Cheeseman, I. M., &#38; Desai, A. A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci. STKE 2005 (266), pl1 (2005).</li>
	<li>Deppert, W. R., &#38; Luka&#265;in, R. Buffers and Additives. Journal of Chromatography Library. 61 (C), 839&#8211;862 (1999).</li>
	<li>Ugwu, S. O., &#38; Apte, S. P. The Effect of Buffers on Protein Conformational Stability. Pharmaceutical Technology. 28 (3), 86&#8211;108 (2004).</li>
	<li>Linke, D. Detergents: an overview. Methods in Enzymology. 463 (34), 603&#8211;617 (2009).</li>
	<li>Zhang, J. Protein-Protein Interactions in Salt Solutions. Protein-Protein Interactions Computational and Experimental Tools. (18), 359&#8211;376 (2012).</li>
	<li>Goldberg, S. Mechanical/physical methods of cell disruption and tissue homogenization. Methods in Molecular Biology. 424 (Chapter 1), 3&#8211;22 (2008).</li>
	<li>Grabski, A. C. Advances in preparation of biological extracts for protein purification. Methods in Enzymology. 463 (C), 285&#8211;303 (2009).</li>
	<li>Dhabaria, A., Cifani, P., Reed, C., Steen, H., &#38; Kentsis, A. A High-Efficiency Cellular Extraction System for Biological Proteomics. J Proteome Res. 14 (8), 3403&#8211;3408 (2015).</li>
	<li>Glatter, T., Ahrn&#233;, E., &#38; Schmidt, A. Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells. J Proteome Res. 15 (2), 679 (2016).</li>
	<li>Zhao, Q.-Q., Yamada, S., &#38; Jimbo, G. The Mechanism and Grinding Limit of Planetary Ball Milling. KONA. 7, 29&#8211;36 (1989).</li>
	<li>Sheng-Yong, L., Qiong-Jing, M., Zheng, P., Xiao-Dong, L., &#38; Jian-Hua, Y. Simulation of ball motion and energy transfer in a planetary ball mill. Chinese Phys. B 21 (7), 078201 (2012).</li>
	<li>Fulton, A. B. How crowded is the cytoplasm? Cell. 30 (2), 345&#8211;347 (1982).</li>
	<li>Ellis, R. J. Macromolecular crowding: obvious but underappreciated. Trends Biochem Sci. 26 (10), 597&#8211;604 (2001).</li>
	<li>Kalkum, M. Using the Retsch MM301 Ball Mill for Cryogenic Disruption of Yeast Cells. 2 City of Hope, (2003).</li>
	<li>Staley, J. Making Whole Cell Extract of Saccharomyces cerevisiae cells using the Retsch MM301 Ball Mill. Retsch GmbH: (2005).</li>
	<li>Bell, A. W., Nilsson, T., Kearney, R. E., &#38; Bergeron, J. J. M. The protein microscope: Incorporating mass spectrometry into cell biology. Nat Methods. 4 (10), 783&#8211;784 (2007).</li>
	<li>Zhao, X., Li, G., &#38; Liang, S. Several affinity tags commonly used in chromatographic purification. J Anal Methods Chem. 2013 (1), 581093&#8211;8 (2013).</li>
	<li>Waugh, D. S. An overview of enzymatic reagents for the removal of affinity tags. 80 (2), 283&#8211;293 (2011).</li>
</ol>

A correction was made to: Protein Complex Affinity Capture from Cryomilled Mammalian Cells. The References section has been updated.

Erratum: Protein Complex Affinity Capture from Cryomilled Mammalian Cells

的呈现过程的一个典型应用是稳定，将获得的利益内源蛋白质复合体的高收率和纯度为interactomic表征1。可以理解的是这两个稳定地和瞬时相关大分子复合物，主要是由蛋白质的动态网络，协调细胞过程2,3。虽然有用于识别蛋白质-蛋白质相互作用的许多实验方法，亲和捕获是用于分离和解剖生理蛋白质复合物4-6的最广泛使用的方法之一。此外，这种技术具有产生的大分子复合物作为物理实体，而不是仅仅在一个读出的数据点的优势;有利的是，所得到的复合物因此可以在附加的下游的生化，酶和结构测定法7-9中的主机使用。所提出的协议已被开发响应于需要映射protei正蛋白相互作用网络，并在其为细胞生物学效应分子的作用表征大分子复合物。它们相对于它们的应用，以在组织培养物中生长的哺乳动物细胞详细，但也同样适用于广泛的给予适当的系统特定的调整生物样品。 亲和捕获的历史可以追溯到二十世纪初与第一免疫亲和层析实验 - 类似于通常所说时下的免疫沉淀（IP） - 由20世纪50年代初出现在文献中。主流利用技术的进一步通过这个世纪之交，到现在10-13开发。不同的细胞和分子生物学研究已经通过在低温下，至少在过去的四十年磨14-19利用细胞的机械破坏;并利用（超）顺磁珠分离分子BEC有青梅在过去的二十年里20越来越普遍。结合这些不同的技术曾协同改善，可以在复杂的蛋白亲和捕获实验1中可以得到的结果，由我们自己生产工作的多种身体和更广泛的研究团体证明7,9,11,18,19,21 -23。支承结果示于图1中。 注意事项以及适用于执行有效的亲和捕获实验考虑的一个集合在参考文献1中找到。通常这种方法是最合适的:（Ⅰ）编目交互件的目标蛋白， 即，使用蛋白质谱（MS）来识别迄今未知copurifying蛋白质（探索性分析）; （II）的测定对于特定相互作用伴侣的存在下， 即用MS或免疫印迹来检测怀疑在蛋白质的特定蛋白或有限集teract与感兴趣（假设检验）的蛋白质;或（III）制备含有用于通过其他技术进一步研究（制备后处理）感兴趣的蛋白质内源性组装的蛋白质复合物。在开始的亲和捕获试验制度之前它具有结合靶蛋白质，典型地针对感兴趣天然靶蛋白或针对附加到的融合蛋白的标签的IP能力的抗体的高品质的亲和试剂是绝对必要的。它也是关键的是具有代替实验读出的适当的方法:一般蛋白染色（如考马斯亮蓝，SYPRO Ruby或银，以下SDS-PAGE），蛋白质印迹，和蛋白质的MS都常用于与亲和捕获结合使用1。所提出的协议利用抗体复合体的磁珠的亲和力网上平台。而亲和介质的功能，可以最初在利用几个实验参数试验进行验证，以得到每个实验应凭经验优化1,11,24最好的结果。该协议被分成三个不同的阶段:（1）冷冻的细胞材料的准备; （2）通过在低温下的固态研磨细胞破裂;和（3）蛋白提取和使用抗体偶联的顺磁珠亲和捕获。

<table><tbody><tr><td>Growth media &amp;amp; cell culturing implements</td><td></td><td></td><td>For producing frozen cells (I)</td></tr><tr><td>500 cm&lt;sup&gt;2&lt;/sup&gt; culture dishes</td><td>Corning</td><td>431110</td><td>For producing frozen cells (I)</td></tr><tr><td>Large beaker (1-2 L)</td><td></td><td></td><td>For producing frozen cells (I)</td></tr><tr><td>Rectangular ice pan&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>13-900-747</td><td>For producing frozen cells (I), &amp;amp; cryomilling (II)</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td></td><td></td><td>For producing frozen cells (I)</td></tr><tr><td>Large cell scrapers</td><td>Fisher Scientific&amp;nbsp;</td><td>08-100-242</td><td>For producing frozen cells (I)</td></tr><tr><td>Liquid nitrogen&amp;nbsp;</td><td></td><td></td><td>For producing frozen cells (I), cryomilling (II), &amp;amp; affinity capture (III)</td></tr><tr><td>50 ml conical tubes</td><td>Corning</td><td>352098</td><td>For producing frozen cells (I), &amp;amp; cryomilling (II)</td></tr><tr><td>20 ml Luer lock syringe</td><td></td><td></td><td>For producing frozen cells (I)</td></tr><tr><td>Leur lock syringe caps</td><td>www.dymax.com</td><td>T11182</td><td>For producing frozen cells (I)</td></tr><tr><td>Refrigerated centrifuge</td><td></td><td></td><td>For producing frozen cells (I)</td></tr><tr><td>50 ml tube rack, 4 position</td><td></td><td></td><td>For producing frozen cells (I), cryomilling (II), &amp;amp; affinity capture (III)</td></tr><tr><td>Styrofoam box</td><td></td><td></td><td>For producing frozen cells (I), cryomilling (II), &amp;amp; affinity capture (III)</td></tr><tr><td>Planetary ball mill, PM 100&amp;nbsp;</td><td>Retsch</td><td>20.540.0001&amp;nbsp;</td><td>For cryomilling (II)</td></tr><tr><td>Stainless steel milling jar, 50 ml&amp;nbsp;</td><td>Retsch</td><td>01.462.0149</td><td>For cryomilling (II)</td></tr><tr><td>Stainless steel milling balls, 20 mm &amp;Oslash;&amp;nbsp;</td><td>Retsch</td><td>05.368.0062</td><td>For cryomilling (II)</td></tr><tr><td>Lid gasket: PFA encapsulated spring energized seal, 2.62 mm 44.63 mm&amp;nbsp;&amp;nbsp;</td><td>www.marcorubber.com</td><td>T1044-2.62x44.63</td><td>For cryomilling (II)</td></tr><tr><td>mini-LN&lt;sub&gt;2&lt;/sub&gt; decanter</td><td>homemade</td><td>see Ref 19</td><td>For cryomilling (II)</td></tr><tr><td>250 ml PTFE jar insulator (optional)</td><td>homemade</td><td>The Rockefeller University</td><td>For cryomilling (II); can be produced by The Rockefeller University High Energy Physics Instrument Shop, or design blueprints provided, upon request</td></tr><tr><td>PTFE puck insulator (optional)</td><td>homemade</td><td>The Rockefeller University</td><td>For cryomilling (II); can be produced by The Rockefeller University High Energy Physics Instrument Shop, or design blueprints provided, upon request.</td></tr><tr><td>5 bar/500 kPa pressure relief valve (optional)</td><td>http://www.leeimh.com/</td><td>558 series check valve</td><td>For cryomilling (II)</td></tr><tr><td>1.5-2 ml microfuge tubes</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>Extractant</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>Complete EDTA free protease inhibitor cocktail tablets</td><td>Roche Applied Science</td><td>11873580001</td><td>For affinity capture (III)</td></tr><tr><td>Vortex mixer</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>Ice bucket</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>Microtip sonicator, Q700</td><td>Qsonica</td><td>Q700</td><td>For affinity capture (III)</td></tr><tr><td>low intensity 1/16&amp;rdquo; microtip probe</td><td>Qsonica</td><td>4717</td><td>For affinity capture (III)</td></tr><tr><td>Bench-top refrigerated microcentrifuge</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>Dynabeads M-270 Epoxy</td><td>Thermo Fisher</td><td>14302D</td><td>For affinity capture (III)</td></tr><tr><td>Antibody or affinity ligand coupled to Dynabeads</td><td>homemade</td><td></td><td>For affinity capture (III); see reference 19</td></tr><tr><td>Sample rotator&amp;nbsp;</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>Neodymium magnet</td><td>Life Technologies</td><td>123-21D&amp;nbsp;</td><td>For affinity capture (III)</td></tr><tr><td>SDS-PAGE sample buffer (no reducing agent)</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>DTT</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>SDS-PAGE system</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>SDS-polyacrylamide gel</td><td></td><td></td><td>For affinity capture (III)</td></tr><tr><td>Protein Stain</td><td></td><td></td><td>For affinity capture (III)</td></tr></tbody></table>

protein complex affinity capture from cryomilled mammalian cells

亲和捕获是隔离的内源性蛋白复合物的进一步研究的有效方法。当在与抗体结合使用，这种技术也经常被称为免疫沉淀。亲和捕获可在实验室规模和中高通量上下文被应用。当与蛋白质质谱偶联，亲和捕获已被证明是相互作用组分析的主力。虽然有许多潜在的方法来执行所涉及的许多步骤，以下协议实现我们的青睐方法。两个功能特色:采用cryomilled细胞粉产生细胞提取物和抗体偶联顺珠亲和力网上平台。在许多情况下，我们已经获得了优异的结果，以与更常规的亲和捕获做法获得的那些。低温球磨避免了与其他形式的细胞破碎的相关联的许多问题。它提供了材料的有效破损，同时避免代纳用加热或发泡关联uration问题。它保留了天然蛋白质浓度高达萃取的点，减轻大分子的离解。它减少了时间提取的蛋白质花费在溶液中，限制有害的酶活性，并且其可以由亲和介质减少蛋白质的非特异性吸附。微米级磁性亲和介质已成为在过去几年越来越普遍，越来越多地取代了传统的琼脂糖和基于琼脂糖的介质。磁介质的主要优点包括通常较低的非特异性蛋白质吸附;没有排阻限制，因为蛋白复合物发生结合珠表面，而不是毛孔内的;并利用磁铁易于操纵和处理。

图1，面板I说明和低温球磨磁珠能共同提高样品的质量。面板11A-C表明，含有用于亲和捕获的不溶介质的材料可以影响所回收的蛋白质组19。感兴趣的蛋白质，纯化的FLAG标记的细菌碱性磷酸酶（BAP）中，掺入人细胞提取物。预计不会BAP以特异性相互作用并copurify人类蛋白质。 copurifying蛋白质，通过SDS-PAGE和考马斯染色判定的签名，根据所使用的介质上变化。微米级磁珠表明最清洁轮廓，指示非特异性蛋白质吸附的相对较低的水平。板ID和IE的说明细胞裂解的方法也可以影响所回收的蛋白质组。在提供的例子中，内源蛋白质复合物（在NEXT络合物37），进行亲和力captu来自重或cyromilled超声细胞提取物19。当细胞提取物从cryomilled细胞粉末生产中观察到较少的高质量的污染物。 用亲和捕获，研究内源性蛋白复合物当A的挑战是，它可能是很难区分的误报（FPS），感兴趣的蛋白质善意的生理干扰作用。的FP可以从许多来源，包括非特异性吸附到管和血管，亲和介质，所述抗体或亲和配体的出现，和/或感兴趣的自身蛋白。其结果是，显著必须努力致力于优化捕获过程。 FP的检测仍然是其中大量不同的工具被开发出来，包括实验38-40和计算方法41-43，各有不同的优点和缺点面临的主要挑战。在我们自己的实验中，我们先前注意到的F模式P蛋白结合，与对照细胞提取物α-FLAG磁介质的温育后获得的，这是由在3XFLAG标记的蛋白的存在下所获得的图案不同。此外，这些的FP可以从培养基中温育释放与3XFLAG肽7。这个观察结果与在没有同源表位的FP的机会结合至α-FLAG互补位一致。因为许多研究使用无标记的&#39;亲本细胞"作为一个模拟控制亲和捕获理解这一背景FP的性质是非常重要的;因此，这些控制可能是不恰当的，以确定与标记蛋白质相互作用的特异性。来确定背景或结合贡献的我们的亲和力介质时抗体互补位被占用没有，我们进行了利用3XFLAGα-FLAG磁性介质和HEK 293T细胞提取物（无标签蛋白表达）的存在或不存在模拟亲和捕获实验肽秒杀项。结果示于图1，面板二。 （的≳10毫克/毫升的总蛋白）含有500mM NaCl和1％体积/体积的Triton X-100的20mM HEPES钠pH 7.4的30分钟简言之，将α-FLAG磁性介质在细胞提取物温育。培养基然后用1mg / ml的3XFLAG肽温育以竞争性置换结合到α-FLAG互补位天然或用SDS-PAGE样品缓冲液来实现变性洗脱任何蛋白质。此外，同样进行实验时，细胞萃取物用1微克/毫升和3XFLAG肽10μg/ ml的加标。所有的洗脱级分进行SDS-PAGE和银染色。我们观察到，在没有其同源表位的α-FLAG介质并捕获可与3XFLAG肽洗脱背景蛋白质的可检测的水平-在由图1-II提供的例子中，在37之间，观察到一个优势种50 kDa的。洗脱模式的SDS-PAGE样品缓冲液是可比，用另外的突出物种。然而，在1微克/毫升3XFLAG肽的存在，FP结合到亲和介质去除到低于检测的水平，并在任一天然或变性洗脱级分没有观察到。这一结果表明，未占用的抗体互补位可以是在没有他们的同源表位的混杂和显著到甚至结合在高亲和力的表位的抗体亲和捕获过程中获得的蛋白质组贡献，因为是为3XFLAG /α-FLAG M2的情况下配对。它还suggestd该非常严格的条件下，使用高盐，通常选择以减轻非特异性结合，可以在不存在抗原/抗体相互作用的效果不佳。后续我们进行的SDS-PAGE和定量MS（ 图S1）的类似实验，包括分析。量化347蛋白，我们观察到，每一个蛋白质保存一（假迪斯科非常率= 1％; 表S1）与3XFLAG标记诱饵蛋白相关联相比于3XFLAG肽掺入对照样品。这些结果表明，在我们的实验中，绝大多数观察蛋白质有可能与带标记的蛋白质以共纯化或者是脱靶副产品未被占用α-FLAG互补位的。根据我们的经验，高信号与噪声的亲和捕获结果在SDS-PAGE和蛋白染色由锐利，丰富，大致计量的乐队，以及来自其他微弱的乐队的背景染色的缺乏的离散模式的例子;这一原则无数例子可以在参照图11中可以看出。 为低温球磨时使用的PTFE绝缘体（推荐）的动机是减少罐的变暖的速度研磨期间，在研磨过程中减轻重复LN 2冷却的负担，以及降低冰的程度形成的罐子。这有利于一致研磨性能并简化了细胞粉的处理。实施例绝缘子可以在参考中找到27（冰球），并在图2中，下方（套筒和-冰球）描绘。 <img alt="图1" src="/files/ftp_upload/54518/54518fig1.jpg" /> 图1:选择有代表性的成果。 （Ⅰ）该面板已从参考19进行修改。考马斯蓝染色的SDS聚丙烯酰胺凝胶。 （左）微米尺度磁珠炫耀目标比基于琼脂糖媒体结合低。 （A）磁珠; （ 二 ）传统的琼脂糖; （C）浸渍铁（磁）琼脂糖;各自耦合到抗FLAG M2抗体和用于捕获FLAG标签细菌碱性磷酸酶（BAP;标记的）掺入从cryomilled HEK-293细胞制备的提取物。基于琼脂糖纯化显示出较高水平的非特异性吸附（未标记带）的。从cryomilled HeLa细胞产生的（右）提取物用于纯化的内源蛋白复合物时在抗体偶联的磁珠比那些通过超声处理产生的具有较低的脱靶吸附。 LAP标记44 RBM7使用连接到多克隆抗GFP抗体磁珠纯化NEXT复杂19,37（D）抓获亲和力提取cryomilled粉生产的;通过完整细胞产生的超声（E）的提取物。垂直黑条突出显示了观察到的高质量的非特异性相互作用物通过超声处理制备的样品中的富集的凝胶的区域。黑色箭头指示的预期特定交互件（标记的）。在车道D和E观察〜50 kDa的乐队是由于美洲驼IgG重链。 （II）的银染色的SDS聚丙烯酰胺凝胶。 （标准）有模拟亲和捕获开展了HEK-293T细胞中提取的标准方式。捕获后，将结合的材料的洗脱通过（N）的非变性用1毫克/毫升3XFLAG肽或（D）变性在SDS-PAGE样品缓冲液中洗脱的洗脱来实现; （PB 1）标准。但3XFLAG肽包括在1微克/毫升的提取液，以阻止亲介质上的α-FLAG互补位; （PB 10），PB 1，但在10微克/毫升3XFLAG肽的存在。 IgG的相关频段和3XFLAG肽标记。 <a href="http://ecsource.jove.com/files/ftp_upload/54518/54518fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54518/54518fig2.jpg" /> 图2:袖和冰球PTFE绝缘体。在250毫升PTFE广口瓶（1）所示。 50毫升的不锈钢研磨罐（2）PTFE罐（3）内符合并提供机会留给周期之间的磨内安装了研磨罐。上部聚四氟乙烯绝缘体冰球（2）的夹紧组件和罐子的盖子之间。以冷却安装罐子和绝缘体组件（3），LN 2直接添加到绝缘体的主体，围绕所述罐。铣削的下一个周期可以被启动。 <a href="http://ecsource.jove.com/files/ftp_upload/54518/54518fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 建议集中 </td><td> 笔记 </td></tr><tr><td>氯化钠</td><td> 0.1-0.5中号</td><td>高浓度（&gt; 300毫米）趋于完善总蛋白提取和保持背景很低，但可能会夺走一些，否则除稳定演员。低于150 mM计的浓度通常不减少非特异性背景有效。 </td></tr><tr><td>醋酸铵</td><td> 0.2-2中号</td><td>的盐，包含两个缓冲区，即产生一个中性pH液57。高浓度稳定一些蛋白质复合物。酸性溶液可以从旧的，储存不当结晶导致股票账户上的氨损失。没有额外的pH缓冲剂或盐是必需的在含乙酸铵萃取。可以用NaCl组合以调制所获得的结果。 </td></tr><tr><td>吐温20 </td><td> 0.1％体积/体积</td><td>非离子表面活性剂58;通常与氯化钠相结合。 </td></tr><tr><td>的Triton X-100 </td><td> 0.5-1％体积/体积</td><td>非离子表面活性剂58;典型地与任一的NaCl或NH 4 CH 3 CO 2 H结合</td></tr><tr><td> CHAPS </td><td> 5毫米</td><td>两性离子洗涤剂58 &lt;/ SUP&gt;;通常与氯化钠相结合。 </td></tr><tr><td>肌氨酸</td><td> 1毫米</td><td>一种阴离子洗涤剂58，降低背景，并且可以剥离稳定，成分复杂，有可能揭示二元连接;通常与氯化钠相结合。 </td></tr><tr><td rowspan="2">三Cl </td><td> 20毫米</td><td rowspan="2"> pK a为8.8，在4℃，8.1在25℃。 </td></tr><tr><td> （pH 8.0）中</td></tr><tr><td rowspan="2"> HEPES娜</td><td> 20毫米</td><td rowspan="2"> pK a为7.8，在4℃下，7.5在25℃。 NaOH或KOH用于pH平衡取决于盐（ 例如，氯化钠，氯化钾，或CH 3 CO 2 K），在萃取剂使用。 </td></tr><tr><td> （pH 7.4）中</td></tr></tbody></table> 表1:纯化蛋白复合物的一些建议试剂。此表l派一些试剂，我们已经找到了从人类细胞系纯化蛋白复合物是有用的，以及建议工作浓度。一个典型的萃取制剂包含pH缓冲液，盐和洗涤剂1,11,24,45-48。最好的做法是，以确定试剂的至少复杂组合产生所期望的结果。 <img alt="图S1" src="/files/ftp_upload/54518/54518figS1.jpg" /> 图S1:混合后误报法（MAP-）SILAC分析。 一示意图:在该实验中3XFLAG标记（从pLD401表示）ORF2p蛋白质复合物从重链和轻标记的HEK-293T细胞7纯化，分别。光标记的细胞提取物与3XFLAG肽飙升，防止竞争性抑制3XFLAG标记ORF2p的亲和捕获;这个预计不会阻止可能发生的非 - 技术规格蛋白质的结合与磁介质或抗体的非paratopic结构美云。从磁珠洗脱后，重链和轻样品混合纯化后和通过定量MS分析（MAP-SILAC 39），以确定与任一样本相关的蛋白质的级分;进一步的方法细节位于表S1。 II。银染色的凝胶示出的轻和重标记的材料得到可比较的结果（比较L的H），和，在3XFLAG尖峰在（LI）的存在下进行的纯化是竞争性抑制。下面，考马斯蓝G-250染色的凝胶塞组成的混合H和LI馏分，基于凝胶的蛋白质组学分析之前，如在表S1说明。 <a href="http://ecsource.jove.com/files/ftp_upload/54518/54518figS1large.jpg" target="_blank">请点击此处下载本图的放大版本。</a> 表S1:MAP-SILAC。此表包含在图S1中所描述的MAP-SILAC实验的执行而得到的数据。 <a href="http://ecsource.jove.com/files/ftp_upload/54518/Table_S1.xlsx" target="_blank">请点击这里下载此表。</a>

Watch this Scientific Journal Video about Protein Complex Affinity Capture from Cryomilled Mammalian Cells at JoVE.com

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

这里，我们描述协议破坏由固态研磨哺乳动物细胞在低温下，产生从所得细胞粉细胞提取物，并且在抗体偶联微米级的顺磁珠亲和捕获隔离感兴趣的蛋白质复合物。

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 ...

protein-complex-affinity-capture-from-cryomilled-mammalian-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

14.9K 次观看.  The Rockefeller University. 这里，我们描述协议破坏由固态研磨哺乳动物细胞在低温下，产生从所得细胞粉细胞提取物，并且在抗体偶联微米级的顺磁珠亲和捕获隔离感兴趣的蛋白质复合物。 

视频: Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

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 ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

遗传学

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

生物学

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the posttranslational modifications, microtubules can form complexes with various interacting proteins. These microtubule-protein complexes are often implicated in human diseases. Understanding the structure of such complexes is useful for elucidating their mechanisms of action and can be studied by cryo-electron microscopy (cryo-EM). To obtain such complexes for structural studies, it is important to extract microtubules containing or lacking specific posttranslational modifications. Here, we describe a simplified protocol to extract endogenous tubulin from genetically modified mammalian cells, involving microtubule polymerization, followed by sedimentation using ultracentrifugation. The extracted tubulin can then be used to prepare cryo-electron microscope grids with microtubules that are bound to a purified microtubule-binding protein of interest. As an example, we demonstrate the extraction of fully tyrosinated microtubules from cell lines engineered to lack the three known tubulin-detyrosinating enzymes. These microtubules are then used to make a protein complex with enzymatically inactive microtubule-associated tubulin detyrosinase on cryo-EM grids.

Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron microscopy.

从哺乳动物细胞中提取修饰的微管以通过冷冻电子显微镜研究微管 - 蛋白质复合物

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

免疫与感染

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 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...