This work was funded by the Wellcome Trust (WT098051).

1. FLAG亲和纯化分离天然蛋白复合物的<ol><li>准备工作<ol><li>设置一个37℃的水浴中解冻细胞沉淀。 </li><li>冷却微量至4℃。 </li><li>放置不同尺寸的几个微量离心管（1.5毫升，15毫升）并在冰中均化。 </li><li>制备10毫升的裂解缓冲液（50mM的Tris-HCl pH 8的，150mM NaCl的，​​0.1％的Nonidet P-40，1mM EDTA）中，并保持在冰上。使用缓冲刚刚之前，加1 M DTT的10μL和无EDTA的蛋白酶抑制剂的压碎片剂。 </li></ol></li><li>的抗体联珠的制备 注:抗体包被的珠可以在预先准备长达一周和直到需要保存在冰箱中。蛋白G具有比A蛋白更高的亲和力对于大多数物种的免疫球蛋白亚型中，除了兔IgG，为此，蛋白A具有更高的亲和力。 <ol><li>洗的50μL蛋白G包被的磁珠:将50＆＃956;珠浆状物到微量离心管中的L，放置管在磁体收集在管侧的珠子和除去液体。重悬珠子于0.5mL PBS-0.01％吐温20的。 </li><li>放置管在磁体收集在管侧的珠子和除去液体。重悬珠子在PBS-0.01％Tween-20的40μL。加入10微克（1毫克10μL/ mL的储备溶液）M2抗FLAG抗体。用在室温下20分钟旋转孵育。 </li><li>放置管在磁体收集在管侧的珠子和除去液体。用0.5mL PBS-0.01％吐温20的洗涤珠。 </li><li>用于交联的抗体与珠，用1mL的0.2M三乙醇胺pH 8.2的的洗珠两次。然后，重悬在1mL 20mM的DMP（二甲基pimelimidate）的小珠在0.2M三乙醇胺pH 8.2的（DMP溶液应准备立即使用前新制）。与旋转在室温下孵育30分钟。 </LI&gt; <li>放置管在磁体。去除上清，于0.5mL的50mM的Tris-HCl pH为7.5的重悬珠子。与旋转在室温下孵育15分钟。 </li><li>用0.5mL PBS-0.1％吐温-20的洗涤珠3次。直到需要留珠最后一次洗涤。 </li></ol></li><li>基于FLAG亲和纯化 注:请注意在没有缓冲不留珠的很长一段时间。所有的缓冲区和管应始终被保存在冰中。以下方案用于蛋白质复合物的2-5×10 8个细胞表达FLAG标记的蛋白20的内源性水平的纯化（10-20毫克蛋白质裂解物）最优化。 <ol><li>解冻在水浴将细胞沉淀在37℃下，直到它开始熔化。采取管从浴，轻轻地旋转，直到沉淀完全解冻，立即将装有在冰上将细胞悬浮液的管中。 </li><li>加入5毫升冰冷的裂解缓冲液（含有DTT和蛋白酶抑制剂），以细胞悬浮液和涡流混合。孵育在冰中10分钟。 </li><li>转移裂解液冷Dounce均匀。裂解用紧杵20-30招（直至没有明显的粘度）。转移匀浆冷离心管。 </li><li>离心机在18000×g下在4℃下15分钟。 </li><li>转移澄清裂解液到一个干净的冷水管。离开裂解液50μL后面。采取裂解物的35μL等分试样来测量蛋白质浓度和监测纯化。 </li><li>从珠中取出PBS-0.1％Tween-20的。重悬珠子用1mL裂解物，并用裂解物的剩余部分混合。孵育在旋转轮上的混合物在4℃下1-2小时。 </li><li>收集与所述磁体的管侧的珠。收集上清液30μL等分试样并弃去上清液的其余部分。重悬珠子于0.5mL IPP150缓冲液（10mM的Tris-HCl pH值8,15的为0mM NaCl的，​​1mM的EDTA，0.1％NP-40）用移液器4-6倍。转移到1.5毫升冷管。 </li><li>用0.5mL IPP150缓冲两次以上的重复洗涤。 </li><li>洗涤用0.5mL FLAG天然洗脱缓冲液（20mM的Bis-Tris pH7的，20mM的氯化钠，0.02％的Nonidet P-40，1mM的EDTA，200mM的ε氨基己酸）的珠三次。随着最后洗脸的时候，珠转移到一个新的冷轧管。彻底清除缓冲区。 </li><li>重悬在100μL200μg/ mL的3×FLAG肽的珠在纯的洗脱缓冲液。孵育在4℃下10分钟并轻轻旋转。 </li><li>收集与所述磁体的珠，并将上清液转移到一个新的冷管。 </li><li>重复步骤1.3.10-1.3.11两次。池中的所有洗脱液。 </li><li>浓缩洗脱液至25μL在离心过滤器单元（10 kDa的标称分子量极限截止，PES）通过以10,000xg离心在4℃下。转移集中洗脱液到一个新的冷管中，加入50％glyceROL的5％的终浓度。将浓缩的洗脱物可在4℃下如果需要，保持过夜。 </li></ol></li></ol> 2.蓝色变性PAGE <ol><li>准备工作<ol><li>准备1升1个原生PAGE阳极缓冲，1个原生PAGE深蓝色阴极缓冲和1个原生Light Blue页阴极缓冲的。 </li><li>除去预制3-12％的天然PAGE凝胶上的梳子和用1X天然PAGE深蓝色阴极缓冲液洗涤所述孔两次。填写与1X天然PAGE深蓝色阴极缓冲井。设置在运行罐凝胶，但不加深蓝阴极缓冲到阴极（内部）室。 </li></ol></li><li>电泳运行<ol><li>准备样品:向25μL浓缩洗脱液分别添加4倍天然样品上样缓冲液的适当体积和0.5％的G-250样品添加剂对于1x最终浓度和0.005％。 </li><li>装载样品和天然分子量标记物留下空孔betweeñ他们。负载10-20μL中的所有井空1个原生样缓冲液中。 </li><li>填用200mL 1×非变性PAGE深蓝色阴极缓冲的仔细的阴极（内侧）腔室，以便不干扰样品。填充550毫升1×非变性PAGE阳极缓冲的阳极（外侧）腔室。 </li><li>运行在150V凝胶30分钟。不停的跑，用血清移液管去除深蓝色阴极缓冲和用1X浅蓝色阴极缓冲更换。持续60分钟运行。所述凝胶可以在室温下或在4℃下运行;温度可能对蛋白质结构复杂的影响。 </li></ol></li><li>凝胶染色<ol><li>打开凝胶盒，并丢弃所述盒板中的一个。凝胶保持连接到另一个盒板。转移凝胶到含有足够固定液（40％甲醇，2％乙酸），以覆盖凝胶孵育轻轻摇动30分钟托盘或容器中。 </li><li>拆下固定SOLUT离子，并添加足够的水，以覆盖凝胶。所述凝胶可以被扫描以供将来参考。 </li></ol></li></ol> 3.质谱分析注意事项:应在层流罩内进行所有后续步骤，如果能够确保样品的清洁度。所有的解决方案应该用HPLC级水中制备。讨论此协议与质谱实验室，将进行分析。 <ol><li>准备工作<ol><li>放置在另一个96孔板的顶部锥形底96孔板收集垃圾。皮尔斯用21号针的锥形底96孔板的孔的底部。 </li><li>洗穿孔96孔板的孔中。 <ol><li>加的50％乙腈-0.25％甲酸200μL到穿孔板的孔中。孵育在摇床的板堆10分钟。 </li><li>离心机在500×g离心1分钟，以使液体流过小孔进入废物收集板。丢弃在WASTE液体。 </li><li>重复步骤3.1.2.1-3.1.2.2两次。 </li><li>填充50mM碳酸氢铵的150μL的刺穿96孔板的孔中（新鲜制备）。 </li></ol></li><li>洗离心过滤96孔平板的孔中。 <ol><li>放置离心过滤板下一个正常的96孔板以收集废物。加的50％乙腈-0.25％甲酸200μL到离心过滤板的每个孔中。 </li><li>离心以200×g的板堆1分钟。丢弃的垃圾。 </li><li>重复步骤3.1.3.1-3.1.3.2两次。 </li></ol></li></ol></li><li>凝胶切除和凝胶内消化 注:虽然切除凝胶，识别并记对准每个蛋白质标准凝胶片。蛋白质标准可以被用来估计近似分子量为所有凝胶切片。 <ol><li>将带有在清洁的表面的玻璃板的凝胶。切片车道含有荷兰国际集团样品放入48个相同切片（1.5毫米×5毫米）的。切割每个切片成2-3更小的块（除了在凝胶的顶部的最后五个切片），并放置各切片顺序地穿孔的一个孔中并洗涤96孔板。 </li><li>添加乙腈的50μL到每个孔中。振摇30-60分钟孵育板。除去离心液体如在步骤3.1.2.2。 </li><li>在50mM碳酸氢铵添加2mM的TCEP的200μL到每个孔中。振荡，在室温下30分钟的孵育板。除去离心液体如在步骤3.1.2.2。 </li><li>在50mM碳酸氢铵加入200μL的4mM碘乙酰胺向每个孔中。与在黑暗中于37℃下振荡30分钟孵育板。除去离心液体如在步骤3.1.2.2。 </li><li>添加50mM碳酸氢铵的150μL乙腈加50μL的和振荡，在室温下30分钟温育该板。重复此洗涤步骤S作为需要，直到所有的蓝颜色从凝胶块除去多次。 </li><li>通过加入纯乙腈的200μL脱水凝胶块，并用在室温下振荡20分钟直至凝胶片是白色的，不透明的孵育。除去乙腈。 </li><li>在冷的50mM碳酸氢铵添加150μL的0.001毫克/胰蛋白酶的溶液（测序级）到每个孔中。在37℃振荡2小时孵育板。 1个小时后，检查凝胶片覆盖有液体;如果不是这种情况下，添加50mM碳酸氢铵的另一50-100μL。在37℃下2个小时后，继续在25℃下消化过夜。 </li><li>将干净的圆锥形底板的含凝胶的板下，从而确保所述板的正确取向。离心收集含有肽以200×g，1分钟的液体。 </li><li>放置肽含板在离心蒸发器和蒸发液体，同时执行吨他下一步。 </li><li>加的50％乙腈-0.25％甲酸150μL到凝胶块。在37℃振荡30分钟孵育。 </li><li>通过离心以200×g，1分钟收集液体到部分干燥的板从步骤3.2.9。继续蒸发含肽板而进行下一步骤。 </li><li>重复步骤3.2.10-3.2.11。 </li><li>添加纯乙腈的200μL含有凝胶片板的每个孔中。在室温下振摇15分钟孵育。 </li><li>离心收集在含肽-板上的液体以200×g，1分钟。 </li><li>确保乙腈的所有孔终浓度为55％以上。如果需要，添加额外的纯乙腈。 </li><li>转移肽溶液至洗涤离心过滤板。将干净的圆锥形底板它下面收集肽。离心以200×g的板堆1分钟。 </li><li>蒸发罐吃了液体在圆锥形底板，直到井完全干燥。该板可以用硅氧烷盖覆盖，并在该阶段储存在-20℃。 </li><li>在0.5％甲酸32μL剧烈振荡15分钟，再溶解肽。添加8μL400 mM碳酸氢铵和剧烈振荡15分钟孵育。离心板以200×g，1分钟。用硅酮盖盖和板保存在-20℃直至质谱分析。 </li></ol></li><li>质谱 注:使用猎枪蛋白质组学方法兼容高分辨率质谱仪分析肽。数据依赖采集是最常用的LC-串联MS建立对于这种类型的分析，并应用于有效肽鉴定被优化，经背景噪声最小化冗余的测序和候选选择。在第一级质量分析扫描（MS1）质谱应记录在简档模式。这是推荐ED优先选择用于第二级质谱分析（MS2）双和三电荷的离子。 M / Z 400-1,600的质量范围是合适的，以确定8-30个氨基酸范围之间最肽。在此，提供了一种使用轨道阱质谱仪进行分析的协议。 <ol><li>注入使用nanoLC-MS / MS系统的自动取样每次分析样品的20μL。脱盐和在反相浓缩肽C18柱（100μm内径2厘米）。 </li><li>使用的4-28％乙腈/含0.1％甲酸30分钟线性梯度，在300升/分钟的分析C18柱（75μm内径，15厘米）分离肽。 </li><li>用具有下列参数的顶部10的数据依赖性获取方法分析在质谱仪的肽:m / z范围380-1,600，在m 30000分辨率/ Z 400，2钍的隔离宽度，在±10ppm的45秒动态排除，自动增益控制目标以1×10 6，用于MS和5000用于MS / MS，最大喷射时间150毫秒为MS和100毫秒MS / MS。 </li></ol></li></ol> 4.数据分析<ol><li>蛋白质鉴定和定量使用MaxQuant <ol><li>使用免费提供MaxQuant软件从质谱原始数据的每个凝胶分数以确定和量化蛋白质。 注:MaxQuant可以工作在数据依赖采集猎枪蛋白质组学方法生成的数据。从蓝色天然凝胶级分的MS数据应在批次作为单独的实验，而不是作为一个实验组合所有凝胶切片的级分进行分析。检查iBAQ值框进行化学计量计算。用于使用MaxQuant数据库搜索和蛋白质定量的详细方案在21中描述了22。 </li></ol></li><li>使用英仙座蛋白质谱分析<ol><li>使用免费提供的软件英仙座显示鉴定蛋白质的偏移剖面，并使用它们进行比较统计分析。 注:关于如何使用英仙座通用文档可在http://www.coxdocs.org/doku.php?id=perseus:user:tutorials。样品数据集可作为补充数据。 </li><li>载由包含所有凝胶分数的蛋白质鉴定和定量值MaxQuant分析返回的proteingroups.txt文件。填充的强度值到主箱（ 图1）。矩阵将出现包含所有蛋白质鉴定中的行，与所述凝胶分数为列。 </li><li>删除对应于扭转命中条目，仅确定了位点和潜在的污染物通过选择基于在过滤行下拉菜单类别列（ 图2）过滤行蛋白质。 </li><li>通过在正常化下拉菜单中选择除，然后萨姆（ 图3）穿过正常化针对总蛋白质强度轮廓的每个蛋白的级分强度。一个新的MATRIX出现。 </li><li>通过在可视化下拉菜单（ 图4）中选择资料显示绘制的所有蛋白质的迁移轮廓图。 </li><li>基于过滤行下拉菜单有效值选择过滤行。在需要有效值的数量输入1。 </li><li>通过在集群/ PCA下拉菜单中选择分层聚类执行所有蓝色天然凝胶分数鉴定蛋白质的层次聚类。取消选择列树盒（ 图5）。簇被可视化在热图的聚类标签中的矩阵（ 图6）旁边。 </li><li>找到包含在树形图诱饵蛋白群集。集群中的其它组件表示蛋白质与诱饵共迁移，且因此相同的复合物的潜在相互作用的蛋白质/亚基。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/55498/55498fig1.jpg" /><b<html> R /&gt; 图1.修斯数据上传窗口的屏幕快照。该图示出了用于加载蛋白质鉴定和定量数据到修斯的步骤。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig1large.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/55498/55498fig2.jpg" /> 图2.截图修斯窗口的对应于条目的污染物的过滤，反向通过位点鉴定的命中和蛋白质。按类别列选择过滤行打开从数据集中消除了选择的各类蛋白质条目的新窗口。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /55498fig3.jpg" /&gt; 图3.修斯正常化窗口的屏幕快照。在标准化菜单中选择鸿沟打开一个新窗口，用不同的选择。选择萨姆除以所有级分蛋白质的总强度在每个级分的蛋白质的强度值。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55498/55498fig4.jpg" /> 图4的屏幕截图示出修斯偏移剖面图。蛋白质可以在下面的轮廓的矩阵以突出显示其相应的简档被选择。工具栏可用于编辑和导出配置文件。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5.截图修斯分级聚类窗口。该图示出了用于使用曼哈顿（L1）的距离度量的蛋白质的分级聚类的设置。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig5large.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/55498/55498fig6.jpg" /> 图6.修斯的屏幕截图示出了树状图和蛋白质强度热图。在主面板的右侧的工作流示出了进行的每个步骤，将所得矩阵，并且可以用来撤消任何步骤。工作流也可以从任何中间矩阵分支。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig6large.jpg" target="_blank">请点击此处查看该F的放大版。</a>igure。 <ol start="3"><li>相对化学计量的计算<ol><li>选择基于他们共同聚类诱饵蛋白的蛋白复合物的亚单位和划定，他们出现在曲线的峰。 </li><li>分别使用由MaxQuant分析返回的iBAQ值为每个亚基中的每个级分，并通过在该部分对应的诱饵iBAQ值正常化。 </li><li>积为诱饵和蛋白复合物亚基的归一化iBAQs在选定峰的级分，并应用线性回归。计算复杂单元的相对量，通过比较它们的标准化iBAQ值的趋势诱饵。 </li></ol></li><li>蛋白复合物的大小估计<ol><li>使用蛋白质标准，以计算预测的分子量为从所述样品通道中的对齐蓝色天然凝胶切片。从这些，通过产生在y轴绘制在x轴凝胶切片和分子量的线性回归模型。 </l我&gt; <li>使用该模型来估计所观察到的分子量范围根据从它们被识别的蓝色天然片（S）的蛋白质复合物（一种或多种）的。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这里，我们描述了使用亲和纯化，然后与定量质谱组合蓝色本地凝胶电泳来解决蛋白质复合物。这种方法提供解开一维蛋白相互作用列表分成功能性蛋白组件的方法。 我们证明基于使用表位标记蛋白的方法。然而，如果表达标记蛋白的细胞系中不可用，另一种可能是使用的抗体针对感兴趣的蛋白质，只要可实现本机在竞争性洗脱的肽。起始材料和珠量的量可能需要根据目标蛋白的表达水平进行修改。我们通常执行该协议与起始原料2-5×10 8个细胞，这是足够的，即使对于具有低表达水平的蛋白质。裂解缓冲液的组合物应当凭经验选择以实现接近完成诱饵的溶解。这可能是某些类型的蛋白质的具有挑战性的，尤其是染色质结合或膜蛋白。替代方案包括增加盐的量，条件是所研究的蛋白复合物是稳定在高盐，或使用超声处理和/或核酸酶处理为染色质结合蛋白20，29。在膜蛋白质的情况下，切换到洗涤剂DDM或毛地黄皂苷是可取30。在的DNA结合蛋白的情况下，有用的是包括这样的核酸酶在纯化步骤20的Benzonase。完全去除的核酸可确保检测到的相互作用蛋白质之间发生，并且不受DNA介导的。 一个关键因素来考虑使用此方法时，正在调查该复合物的稳定性。该过程是漫长的，可能涉及preservin克蛋白质复合物过夜。我们已经取得了两个染色质重塑复合物（D·博德和M·帕多，数据未显示）很成功，但这应该进行评估。如果所需要的亲和纯化步骤可以被缩短。 替代性技术，让本地分馏，如尺寸排阻色谱法，已被广泛应用于超过50年来表征蛋白质复合物。我们和其他人已经表明，蓝色天然PAGE的分辨率优于使用尺寸排阻色谱法20，31，32，33来实现的。蓝色天然PAGE的另一个优点是，它不需要色谱系统，这是昂贵的，而是使用蛋白电泳设备是在实验室普遍。在方面动手的时候，这种方法不涉及比传统GELC-MS / MS更工作接近角或离线色谱分离。然而，由于大多数分级技术做的，它有一个限制它们的分辨率。配合是非常均匀的或接近的质量和形状可以是超越蓝色天然PAGE所提供的分辨率，因此，问题报告可能不会在解决与共享亚基不同复合普遍成功的协议。令人鼓舞的是，我们在分离两个非常相似的四聚体复合分享三个亚基（M·帕多，手稿准备）是成功的。 由于质谱分析已经变得越来越敏感，非特异性相互作用物和污染物甚至微量可以AP-MS样品中被检测到。这里介绍的方法可能通过集中关注与移民峰，在分子量比更高的诱饵迁移峰重合蛋白质从亲和纯化背景污染实际作用因子的歧视帮助单体蛋白质。 一些研究小组已经使用分级分离技术，随后蛋白相关性分析以描绘在细胞水平的蛋白质复合物，而无需先前的隔离10，11，12，34。然而，这可能导致无法检测到亚化学计量的相互作用。通过亲和纯化富集步骤的引入可以帮助克服这一点。这里所描述的方法应该是探索蛋白质复合物的拓扑和解开多个复合给定蛋白质参与在相同的细胞环境中通常是有用的。该战略很简单，适于可能没有昂贵的色谱分离设备解决蛋白质复合物的实验室。

在细胞中，大多数蛋白质通过暂时性蛋白质 - 蛋白质相互作用或通过形成稳定的蛋白质组合件执行它们的功能。表征蛋白质相互作用，对于全面理解细胞过程的关键。与质谱法（AP-MS）组合的亲和纯化是最普遍应用的策略，以确定天然蛋白质相互作用之一。在过去十年中取得了仪器功能显著进步使得这种方法非常强大。需要注意的是通过AP-MS实验确定的相互作用包括诱饵和猎物之间的直接和间接关联的混合物是很重要的。此外，经常蛋白质参与相同的细胞范围内几个不同的复合物，其可能具有不同的生物学作用，并因此由AP-MS鉴定的相互作用因子可能代表的不同的蛋白质的组件或功能实体的混合。这是不可能推导这种拓扑信息由简单的AP-MS实验中产生的蛋白质的单维列表先验 。然而，该技术可以进一步利用通过将其与一种或多种方法组合以解决这些组件来定义的蛋白质复合物的结构。 为了解决通过AP-MS鉴定蛋白质相互作用的拓扑结构，几种策略已经被应用。一种方法是使用执行在先前轮实验作为诱饵1中确定的猎物迭代AP-MS实验。虽然信息量很大，这是一个相当劳动密集型任务实验与分析。蛋白的交联在与质谱结合越来越多地被用来推导对蛋白复合物2，3，4，5的拓扑信息。但是，计算中交联肽的最终分析仍是一项艰巨的任务，因此在工作流程中的瓶颈。 MS-裂解的交联试剂的出现应推动有紧密接近中相互作用蛋白6,7的氨基酸残基的映射。另一种方法是亲和纯化与现有的正交分离技术8,9结合。通过凝胶过滤或离子交换，或蔗糖梯度分级色谱分离最近也被与定量质谱组合使用在一个系统范围内的电平，以描述多蛋白复合物，通过绕过复杂的分离步骤10，11，12，13。蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）已被广泛地应用到调查天然蛋白的相互作用，通常是那些涉及线粒体膜蛋白复合物14。这种分离技术最近还结合使用无标记蛋白定量和相关性分析，不仅对线粒体复合物15，16，17，但也可用于从全细胞18,19解开其他蛋白复合物。我们假设，亲和纯化的，随后分馏天然方法和定量MS的组合应提供用于解决含有特定蛋白多蛋白组件的有用策略。 在这里，我们描述了结合了通用的基于表位的亲和纯化与分离的复合物的蓝色非变性聚丙烯酰胺凝胶电泳的方法，随后定量质谱属ectrometry和蛋白质的相关性分析，解决了多个组件的蛋白可能参与。我们采用其中融合到表位标签的目的蛋白从内源性基因表达来实现接近生理丰度和确保有效的天然小鼠胚胎干细胞复杂的隔离。这种方法的揭开多种相互作用的蛋白质接合，解决这些基于其相关波形不同组件，而无需比常规GELC-MS 20更多的工作。

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Protein G Dynabeads</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">10003D</td>
			<td style="width:452px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">FLAG antibody M2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Tween-20, protein grade, 10% solution</td>
			<td style="width:285px;">Millipore</td>
			<td style="width:220px;">655206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Dimethyl pimelimidate</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">80490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">0.2 M Triethanolamine buffer, pH 8.2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">T0449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">3x FLAG peptide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Vivaspin 5K NMWCO, PES</td>
			<td style="width:285px;">Sartorius</td>
			<td style="width:220px;">VS0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativePAGE 3-12% Bis-Tris gel</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Running Buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Cathode Additive</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">4x NativePAGE sample loading buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativeMARK</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">LC0725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NativePAGE 5% G-250 sample additive&#160;</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc conical bottom 96-well plate, polypropylene</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">249944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Multiscreen HTS 96-well filtration system</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">MSDVN6550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc 96-well cap natural</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">276002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Iodoacetamide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">I1149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Acetonitrile, HPLC grade</td>
			<td style="width:285px;">Fisher Scientific</td>
			<td style="width:220px;">A/0627/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Trypsin, sequencing grade</td>
			<td style="width:285px;">Roche</td>
			<td style="width:220px;">11418475001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Software</td>
			<td style="width:285px;"></td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MaxQuant (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111795/maxquant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Perseus (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111810/perseus</td>
		</tr>
	</tbody>
</table>

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.

通过质谱法的亲和纯化蓝天然蛋白相关性分析的工作流（ABC-MS）策略在图7中描述。所关注的蛋白质周围天然蛋白质复合物通过使用抗表位标签的亲和纯化（在这种情况下FLAG）和竞争性洗脱分离。该复合物通过BN-PAGE分离，和整个凝胶泳道被切除成48个部分，并且对猎枪LC-MS / MS制备。定量MS信息被用来产生用于穿过蓝色天然分离每个所识别的蛋白质的迁移分布。该相互作用的蛋白质，以形成具有重叠的峰的独特复合物显示相似的迁移曲线。当感兴趣的蛋白参与一个以上的组件中，多个峰值在其偏移剖面观察到的，给定的子复合物是蓝色天然凝胶的分辨能力之内。米格系统比较配给轮廓可以通过蛋白相关性使用分级聚类谱来实现。树状图和所有级分的峰强度被可视化在热图，促进相互作用属于不同的蛋白质复合物的蛋白质（ 图8）的识别。 我们用这个策略来分析MTA2，在体NuRD质重塑复合体20的核心亚基的互动合作伙伴。 如图9所示，MTA2的迁移分布显示700 kDa的和1.2 MDA之间不同强度的两个峰值，用显示高丰度较低的质量峰。其他体NuRD核心亚基，包括MTA1 / 3，中Hdac1 / 2和MBD3，显示完全相同分离模式，尽管峰为一些子单元，即Chd4的，Gatad2a / B和RBBP4 / 7，具有逆多度分布（ 图9A， B）。相比之下，CDK的轮廓2ap1，该招募NuRD复合对Wnt基因的启动子23的调控因子，只显示在较高的质量峰（ 图9C），所以没有Sall4中，这也被证明结合体NuRD 20，24，25转录阻遏。因此，通过蓝色天然PAGE亲和纯化的MTA2相关蛋白的分级分离能解决两种不同形式的NuRD复合的。 ABC-MS还允许新的交互件的标识，同时将其分配给特定的蛋白质实体。通过在一个热图表示的蛋白簇和分数强度的检查，我们检测体NuRD亚基和WDR5，所述MLL甲基络合物26的调节亚基，与WDR5显示与所述两个体NuRD p重合两种迁移峰之间的强相关性eaks（ 图8），表明WDR5和体NuRD之间的新颖的相互作用。我们证实了在体积排阻色谱20免疫共沉淀和共迁移这种相互作用。 距离度量的计算中的分级聚类的选择将影响簇并因此相关的形状。我们建议对不同的指标进行试验，实现了与关注的蛋白质或复杂的现有知识的相互作用的最佳选择。一般来说，我们取得了最好的结果与曼哈顿（L1）的距离度量20。 Pearson相关或欧几里得距离度量也有报道用于基于替代分级分离技术10，11，12配合。 该全从蓝色天然馏分得到titative MS数据也可以被用来确定蛋白质复合物的化学计量。所述iBAQ（基于强度的绝对定量）值提供了鉴定的蛋白27，28的相对丰度的量度。用于在迁移轮廓峰内的所有级分中的蛋白复合物成员iBAQ值是归一化到所述诱饵蛋白，以获得相对量。跨越轮廓峰的归一化iBAQs对于给定的蛋白质复合物构件应该遵循水平趋势，而趋势值反映相对于诱饵蛋白的相互作用蛋白的化学计量。对于化学计量计算的更详细的表示，看20。 <img alt="图7" src="/files/ftp_upload/55498/55498fig7.jpg" /> 图7:示意性的工作流程总结ABC-斯小姐战略研究。该图是从20修改。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/55498/55498fig8.jpg" /> 图8的MTA2相互作用物的BN-PAGE迁移谱分级聚类。基于其移民曲线的相似MTA2相关蛋白呈簇状。仅含有NuRD复合的热图的子集被示出（封闭在蓝色框）。黄色框突出WDR5与NuRD复合体的强相关性。带注释的分子量是从在同一凝胶上运行蛋白标准迁移距离估计。这项研究最初发表在分子与细胞蛋白质组学20的美国生化和Mo<html> lecular生物学。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/55498/55498fig9.jpg" /> 图9.体NuRD亚基和相关蛋白的BN-PAGE迁移轮廓。 A）存在于一个类似的强度图案和MTA2亚基体NuRD。 B）MTA2和体NuRD亚基与逆强度图案。 C）MTA2和CDK2AP1，这是目前仅在较高分子量体NuRD实体。带注释的分子量是从的蛋白质标准的迁移分布估计。这项研究最初发表在分子与细胞蛋白质组学20美国社会的生物化学与分子生物学。_blank"&gt;请点击此处查看该图的放大版本。 <img alt="补充文件" src="/files/ftp_upload/55498/proteinGroupsDB04.jpg" /> 补充数据:样本数据集。 MaxQuant衍生的含蛋白质的鉴定和定量值从ABC-MS实验proteingroups.txt文件。这项研究最初发表在分子与细胞蛋白质组学20美国社会的生物化学与分子生物学。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/proteinGroupsDB04.txt" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling at JoVE.com

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

这里，我们提出使用无标记的定量质谱法对蛋白复合物的亲和纯化和由蓝色天然PAGE它们的分离协议，其次是蛋白相关性分析。这种方法是非常有用的解决相互作用组分成不同的蛋白复合物。

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 ...

resolving-affinity-purified-protein-complexes-blue-native-page

Nanyang Technological University

Research

JoVE Journal

Biochemistry

13.7K 次观看.  Wellcome Trust Sanger Institute. 这里，我们提出使用无标记的定量质谱法对蛋白复合物的亲和纯化和由蓝色天然PAGE它们的分离协议，其次是蛋白相关性分析。这种方法是非常有用的解决相互作用组分成不同的蛋白复合物。 

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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

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

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

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

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

科研

生物化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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

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

从真核细胞蛋白质复合体串联亲和纯化

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

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

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

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

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

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

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

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

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

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

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

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

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

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

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

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

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

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...