Work in the Ait-Si-Ali lab was supported by the Association Fran&#231;aise contre les Myopathies T&#233;l&#233;thon (AFM-T&#233;l&#233;thon); Institut National du Cancer (INCa); Agence Nationale de la Recherche (ANR), Fondation Association pour la Recherche sur le Cancer (Fondation ARC); Groupement des Entreprises Fran&#231;aises pour la Lutte contre le Cancer (GEFLUC); CNRS; Universit&#233; Paris Diderot and the ''Who Am I?'' Laboratory of Excellence #ANR-11- LABX-0071 funded by the French Government through its ''Investments for the Future'' program operated by the ANR under grant #ANR-11-IDEX-0005-01. EB was supported by an INCa grant.

1.接种HeLa-S3核盐可提取与染色质结合组分的制备<ol><li>细胞的集合<ol><li>在Dulbecco氏改良Eagle培养基（DMEM）中补充有10％胎牛血清，100U / ml青霉素和100μg/ ml链霉素（生长生长的HeLa-S3标示-HA-MyoD的和HeLa-S3标示-HA（对照细胞系）培养基），在37℃和5％CO 2在潮湿的培养箱中。 注意:在使用中描述的协议有可能成为建立的HeLa-S3稳定表达的Flag-HA-的MyoD和HeLa-S3空载体（HeLa细胞-S3标志-HA）转染细胞:19,20，或由作者提供。 </li><li>细胞扩增到每个细胞系（HeLa细胞-S3标志-HA-的MyoD和HeLa-S3标志-HA细胞）的10完全融合150毫米菜肴。 </li><li>收集上清（含非贴壁细胞亚群）为5升微调。 </li><li>用每150毫米盘10mL的PBS洗涤附着的亚群，trypsinize细胞在室温统1分钟023（0.05％胰蛋白酶-EDTA溶液，加入2ml为15厘米的培养皿）。 </li><li>添加4毫升每150毫米盘介质和收集细胞到50ml管中。 </li><li>从步骤1.1.3（在5升旋转器），并从步骤1.1.5细胞结合上清，加入500毫升新鲜的预温热的生长培养基中的每个细胞系。 </li><li>转移细胞悬液至5升旋转器和在潮湿培养箱中于37℃和5％CO 2培养细胞为至少60小时。 </li><li>加入500毫升新鲜生长培养基和生长的细胞24小时。 </li><li>添加1升的新鲜生长培养基和生长的细胞24小时。 </li><li>取出将1ml细胞悬浮液计数细胞并检查细胞存活力。彩色细胞悬液30微升用血球0.4％台盼蓝的30微升和计数活（不是蓝色）细胞在显微镜下。 </li><li> ;可以通过每日加入新鲜生长培养基6×10 5个细胞/ ml -保持细胞密度在2不超过1×10 6个细胞/ ml。最大值量一5升微调为2.5 L. 注意:对于下列步骤，由第2批进行- 2.5升培养物的密度为1×10 6个细胞/ ml。重复步骤1.1.12 - 1.1.14收集约20L （≈20克干粒料）每个细胞系在进行下一步之前。 </li><li>沉淀细胞，在500×g离心5分钟，离心在4℃下，并丢弃上清液。 </li><li>悬浮细胞，通过移液和离心机在500×g离心5分钟100毫升冰冷的PBS中于4℃以洗涤沉淀。 </li><li>通过重悬细胞用25ml冰冷的PBS中，将悬浮液转移至50ml管中并在4℃下以500 xg离心离心5分钟重复洗涤。 注:细胞沉淀可立即使用或被在液氮中冷冻并储存在-80℃下数天。 </li></ol></li><li>细胞核的分离 注:执行步骤1.2.1 - 1.4.3在寒冷的房间。 <ol><li>预寒意buffeRS和均质。 </li><li>如果使用冷冻物质，迅速在37℃下解冻水浴细胞沉淀。 </li><li>悬浮20克在20毫升细胞（≈20ml）中通过，用10毫升缓冲液中在第一，然后加入5毫升，然后加入低渗缓冲液A（ 表3）的（等于细胞沉淀的尺寸的体积）另外5毫升用新鲜的缓冲液洗井吸液管以获得最大的细胞。 </li><li>转印40毫升细胞悬浮到预冷却的均化用紧身杵。 </li><li>均质在均化器20次（在进出动作20）细胞和转移细胞悬液至50ml管中。 </li><li>洗均化用7ml蔗糖缓冲液（初始小区体积的1/3， 表3）中，回收的最大单元。快速传送该悬浮进50毫升管从步骤1.2.5保存细胞核和限制渗漏。 </li><li>染色30微升等分0.4％TR的30微升ypan蓝色，在显微镜下分析来控制裂解效率（如果裂解成功，所有的核应蓝色）。如有必要，重复同质化的一步。 </li><li>离心机以10,000 xg离心7分钟，4℃以沉淀细胞核。保存上清液作为细胞质级分。 </li></ol></li><li>核盐提取（SE）级分的制备<ol><li>重悬细胞核沉淀在8ml蔗糖缓冲液（等于细胞核沉淀的尺寸的体积）。通过加入一滴一滴而在涡流8毫升高盐缓冲液（1细胞核沉淀体积， 表3）的充分混合以获得300mM NaCl的终浓度。孵育在冰上30分钟，每5分钟混合。 </li><li>通过加入蔗糖缓冲液8毫升（总体积的1/3）下降NaCl浓度至150mM的。离心机以13,000 xg离心10分钟，4℃。收集上清（核盐萃取物，SE）。 注:要么继续执行步骤1.3.3或离开SE制备染色质结合的级分，然后超离心两馏分同时期间在冰上级分。 </li><li>超离心机SE在4℃下以85000 xg离心30分钟。取上清（≈26ml）清洗。 </li><li>冷冻在液氮中100μl等分的。继续执行步骤2"蛋白质复合体净化"，使用提取的其余部分。 注意:等分试样被用作步骤5.1.1期间的输入控制。 </li></ol></li><li>染色质结合态的制备<ol><li>重悬沉淀（从步骤1.3.5）。精心在7ml蔗糖缓冲液（等于粒料的大小的体积）。 </li><li>添加氯化钙2至1mM的终浓度（28微升的0.5M 的 CaCl 2 14 ml悬液）来激活MNase（步骤1.4.4）并混合。 </li><li>预热悬浮液在37℃1分钟。 </li><li>加入70微升微球菌核酸（MNase）从0.5获得0.0025 U /μL（1/200稀释终浓度U /μL股票），混匀。 </li><li>孵育在37℃下正好12分钟。混合每4分钟。 </li><li>立即置于冰上反应。 </li><li>停止MNase活性，添加112微升的0.5M EDTA pH 8.0的（1/125稀释）的4 1mM的终浓度。孵育在冰上5分钟。 </li><li>超声处理5次在高振幅1分钟，两人之间的超声处理允许1分钟（10分钟计）休息。 </li><li>超速离心机在85000 xg离心30分钟，4℃。 </li><li>收集上清（富含核分数，NE，≈12ml）中。 </li><li>冷冻在液氮中50微升等分。继续执行步骤2"蛋白质复合体净化"，使用提取的其余部分。 注意:等分试样被用作步骤5.1.1期间的输入控制。 </li></ol></li></ol> 2.蛋白质复合物纯化注:进中从每个细胞系（HeLa细胞-S3标示-HA-MyoD的和控制，HEL平行提取A-S3标志-HA）。 <ol><li>基于升旗净化<ol><li>预洗旗树脂。使用标志树脂的600微升（从商用50％股票）每个实验点（SE和NE馏分对于每个细胞系）。 </li><li>转印树脂成15ml试管，重悬在13ml冷TEGN（ 表3），离心2分钟以1000 xg离心并去除上清。重复5次。在最后一次洗涤悬浮标示树脂分成相等的体积TEGN（300微升每个实验点）之后。 </li><li>每个实验点，混合（分别在15ml试管为12毫升NE和在50ml管中的26毫升SE馏分，）洗涤标示树脂的600微升和相应的蛋白质提取物。孵育在4℃下旋转轮过夜。 </li><li>离心机在4℃，1000 XG 2分钟，抛开上清液，并保持在4℃，直至净化效率试验（2.2）的结果。 </li><li>对于SE的样品，重悬旗树脂在4ml TEGN和转移到15毫升管。重复此步骤3次，每次转印到相同的15ml试管，以避免失去珠。在1000 xg离心和4℃并除去上清液离心2分钟。 </li><li>洗涤标示树脂中使用13毫升TEGN中，并在1000 xg离心，4℃离心2分钟15毫升管7倍。 </li><li>最后一次洗涤，悬浮在1毫升TEGN和转让至1.5 ml后低结合管。在1000 xg离心和4℃并除去上清液离心2分钟。 </li><li>重悬在4毫克/毫升200微升标记肽溶液在每个实验点pH 7.8。混合珠和肽。加入200μlTEGN缓冲液中并在4℃孵育至少4小时或过夜，以洗脱结合的蛋白质。 </li><li>在1000 xg离心离心2分钟，在4℃并转移在树脂和上清液（标示洗脱液），以放置在一个2毫升管的空旋转柱。 </li><li>离心柱，收集遗留在2 ml管上清。 </l我&gt; <li>通过加入TEGN缓冲器到列收集旗树脂并进行用200μl的标记肽溶液（4毫克/毫升）的第二标志洗脱在4℃过夜每个实验点。 </li><li>重复步骤2.1.9 - 2.1.10收集第二次洗脱。结合的第一和第二洗脱液。 </li></ol></li><li>净化效果测试 注:在步2.1.11 - 2.1.12，进行SDS-PAGE和银染色（2.2.1 - 2.2.2）。 <ol><li>取15微升洗脱液，加4倍样缓冲液的5微升和2μl10X还原剂，煮沸5分钟，在96℃下并运行SDS-聚丙烯酰胺4 - 根据制造商的说明12％的凝胶。 </li><li>染色使用银染色试剂盒凝胶（按照制造商的说明）。如果有标志-HA-的MyoD和标志-HA之间的蛋白质模式明显不同模拟洗脱液，特别是标志-HA的MyoD的带（约50 kDa的， 图1A），进入下一个STE页。 </li></ol></li><li>血凝素（HA）为基础的分离纯化<ol><li>洗涤的HA树脂通过转移到15ml试管中，在13ml TEGN的重悬，并在4℃下以1000 xg离心离心2分钟。重复洗涤步骤5次。后在相同体积TEGN缓冲器的最后洗涤悬浮珠。 注意:体积必须根据基于旗纯化效率进行调整。开始从商业50％的股票300微升每个实验点。 </li><li>转移HA树脂每个实验点至1.5 ml低结合管。离心2分钟，在4℃下1000 xg离心，消除了最大的洗涤缓冲液（ 表3）的和从基于旗纯化添加洗脱液至HA树脂。 </li><li>孵育在4℃下旋转轮过夜管。 </li><li>离心机在4℃，1000 XG 2分钟，抛开上清液，并保持在4℃，直至净化效率测试的结果（2.3.12）。 </li><li>回覆暂停在0.5ml TEGN的HA树脂，转移到一个新的低结合1.5mL管中。重复再悬浮一次加入到同为1.5毫升管，以避免在1000 xg离心和4℃失去珠离心2分钟。除去上清液。 </li><li>通过用1ml TEGN的每一次，离心以1000 xg离心2分钟，4℃，并除去上清液（避免丢失珠）洗涤珠8倍。最后一次洗涤后，转移到珠0.5毫升管。 </li><li>除去尽可能多的上清液越好。在pH 7.8珠粒以洗脱结合蛋白加入100微升的HA游离肽溶液（4毫克/毫升）。孵育在4℃下旋转轮过夜。 注意:需要的HA肽的量取决于所充满的复合物来进行调整。 </li><li>在1000 xg离心离心2分钟，在4℃和树脂和上清液（HA洗脱物）转移到放置在2毫升管的空旋转柱。 </li><li>离心柱收集遗留的上清液2 ml管。 </LI&gt; <li>执行用100μlHA肽（4毫克/毫升）的每个实验点的第二洗脱。孵育在4℃下旋转轮过夜。重复步骤2.3.8 - 2.3.9收集第二次洗脱。 </li><li>结合的第一和第二洗脱液。 </li><li>通过SDS-PAGE和银染色试验净化效率（见步骤2.2）（ 图1A）。 </li><li>集中的HA洗脱物，使用离心过滤器单元具有根据制造商的说明10 kDa截止的（标称分子量极限，NMWL）30微升。 </li><li>划分浓缩样品分为两部分:22.5（3/4）和7.5（1/4）微升。卡扣冷冻在液氮中并存储样本的1/4在-80℃。使用3/4部分用于步骤3。 注意:1/4冷冻部分是用于通过免疫印迹质谱结果的确认（参见步骤5.1）。 </li></ol></li></ol> 3.质谱分析注:以下步骤应与质谱设施，将进行分析讨论。 <ol><li>补充样品3/4（22.5微升），4倍样缓冲液7.5微升和3.3微升10X还原剂中，并在96℃煮沸5分钟。 </li><li> 12％的凝胶 - SDS-聚丙烯酰胺4上负荷的样品。在150V恒定5分钟运行凝胶，以允许所有的蛋白质不经分离进入凝胶。 </li><li>用水冲洗凝胶;定为20 - 30分钟用固定液（10％乙酸，50％甲醇，40％的超洁净的水），然后洗固定凝胶5次在超洁净的水。 </li><li>切含有蛋白质，贮存于1ml水的频带在1.5ml管中，发送到质谱设施。 </li></ol> 4.数据分析 注意:这是用于分析的一般指导。确切的步骤，将取决于被MS设施提供的数据的特定模式用于执行分析（ 例如，21,22）。 <ol><li>比较确定的蛋白在与对应阴性对照制剂获得的名单"的MyoD"洗脱液名单。从分析中排除两个列表中发现的蛋白质。 </li><li>删除具有剩余的列表中标识的肽≤2总数的蛋白质。 </li><li>访问功能注释和所获得的使用DAVID生物信息学资源蛋白质的列表的功能分类（http://david.abcc.ncifcrf.gov/home.jsp）和分类 ​​蛋白23,24的列表。 </li><li> （可选）从列表中选择"搭便车"，收取无核蛋白质具有很强的偶然结合DNA 25负电荷删除。这些含有细胞骨架，细胞质，线粒体，膜和受体蛋白质（蛋白质折叠，在表1骨架和杂项组蛋白和2）。&lt;/ LI&gt; <li>比较所得到的由SE和网元级分的MyoD交互件的列表，以确定特定于每个级分（ 图2）共同的蛋白质和蛋白质。 </li></ol> 5.确认通过Western blot鉴定交互件<ol><li>确认在HeLa-S3标志-HA-的MyoD和HeLa-S3标志-HA <ol><li>在冰上步骤2.3.14（7.5微升）获得解冻洗脱液样品。添加12微升TEGN缓冲液，10微升4倍装载缓冲液中，10×3微升还原剂。煮沸样品在96℃5分钟。 </li><li>准备输入样本的印迹。 <ol><li>从步骤1.3.9和1.4.11解冻等分试样并用BCA试剂盒（按照制造商的说明）测量蛋白浓度，例如。 </li><li>使用15微克每车道的蛋白质。测量提取物的合适量和调整样品的体积达到15微升与TEGN缓冲器。 </li><li>加入5微升4倍样缓冲液和1.5微升10X还原剂。煮沸样品在96℃5分钟。 </li></ol></li><li>执行与感兴趣抗体（特异于MS分析过程中确定伙伴候补）使用15微升洗脱液样品的免疫印迹分析。使用输入提取物作为内源性蛋白的水平（ 图3A）的控制。 </li></ol></li><li>确认在相关蜂窝系统<ol><li> C2C12成肌细胞的鼠标总核提取物的制备。 <ol><li>在潮湿的培养箱中生长C2C12小鼠成肌细胞在补充有15％胎牛血清的DMEM培养基，100U / ml青霉素和100μg/ ml链霉素，在37℃和5％的CO 2。不要超过80％的细胞汇合保持增殖状态的细胞。 </li><li>生长C2C12的至少两个150毫米菜肴进行免疫沉淀的一个点（一种抗体）（IP）。吸出介质，用10毫升的PBS洗涤细胞两次。 </li><li>刮细胞和收进15ml试管。铈ntrifuge在250 xg离心在4℃下5分钟。弃上清。 </li><li>估计细胞沉淀（= V 细胞 ）的容积。轻轻悬浮在低渗的缓冲液B 3体积V CELL粒料破坏细胞质膜（ 见表3）。 </li><li>添加10％0.44 x垂直细胞体积的NP-40，以达到1％的最终浓度（体积/体积）。轻轻颠倒管数次​​混匀。 </li><li>添加的SR（ 表3）的0.89 x垂直细胞体积，以保持核的完整性。轻轻颠倒试管几次以均匀的悬浮液，并离心5分钟，在2000 xg离心以沉淀细胞核。 </li><li>除去上清液（细胞质分数）。估计细胞核沉淀（= V NUC）的体积。重悬细胞核沉淀在蔗糖缓冲液的一个体积V NUC。 </li><li>通过加入一滴一滴而在高盐缓冲液的旋涡1体积V NUC充分混合以获得300mM的终浓度NaCl和孵育在冰上30分钟。混合每5分钟。 </li><li>添加蔗糖缓冲液中的一个体积V NUC（降低NaCl浓度至150毫摩尔）和氯化钙2（最终浓度为1mM）。混合。 </li><li>预热悬浮体在37℃下1分钟，加入微球菌核酸至终浓度0.0025 U /μL（1/200 0.5 U /μL的股票）的。混合。 </li><li>孵育在37℃下正好12分钟。混合每3分钟。 </li><li>立即置于冰上反应，并添加EDTA（终浓度为4毫米）停止MNase活动。孵育在冰上5分钟。 </li><li>超声处理5次在高振幅每次0.5分钟。 2超声处理之间，允许0.5分钟（5分钟计）休息。 </li><li>超离心机在85000 xg离心30分钟，4℃。收集上清（总核提取物）。 </li><li>用BCA试剂盒（按照制造商的说明）测量总核提取物的蛋白质浓度，例如并继续immediat伊利步骤5.2.2。 </li></ol></li><li>内源性的MyoD的免疫沉淀（IP）。 <ol><li>要预清提取物，洗10微升的G蛋白琼脂糖珠为每个IP点。 <ol><li>转移的G蛋白琼脂糖珠的所需量到1.5毫升管，悬浮1.4毫升冷TEGN，离心机中2分钟，在1800 xg离心并去除上清。重复3次。 </li></ol></li><li>混合预洗涤G蛋白琼脂糖珠与总核提取物的合适体积。使用500微克每IP的总点数核提取蛋白质。 </li><li>孵育在旋转轮在4℃下2小时进行预清除提取物。 </li><li>离心机在1800 xg离心在4℃下5分钟。保留上清。 </li><li>混合的MyoD抗体的5微克或5微克兔IgG与在1.5ml低结合管500微克预清总核提取物。添加TEGN最多缓冲1毫升在4℃孵育的旋转轮过夜。 注意:请在F5.2.2.3一步期间ollowing步骤。 </li><li>每个IP点预洗7.5微升蛋白质A / G股珠的解决方案。 <ol><li>在1800 xg离心转移的珠的所需量到1.5毫升管，悬浮珠在1ml TEGN和离心机在4℃下2分钟。去除上清。重复3次。 </li></ol></li><li>重悬蛋白质A / G珠在1.5ml于4℃过夜封闭溶液（ 表3），并培育上的旋转轮。 </li><li>与16,000 XG在4℃下10分钟，ABS（步5.2.2.5）培养离心机总核提取物。保留上清。 </li><li>离心机在1800 XG阻止蛋白A / G树脂（步骤5.2.2.7），2分钟，4℃。弃上清，悬浮在1毫升TEGN缓冲。重新离心在4℃下1800×g离心2分钟洗出封闭缓冲液。 </li><li>悬浮阻断蛋白质A / G的树脂在1ml TEGN缓冲和分装成新的低蛋白结合管，以获得7.5微升一个BLO每个IP点cked蛋白A / G树脂。离心机在1800 xg离心在4℃下进行2分钟，并除去尽可能多的上清液。 </li><li>添加用绝对温育总核提取物（或对照IgG）与A / G树脂。混合并孵育在旋转轮上室温（RT）2小时。 </li><li>离心悬浮液在RT 1800 xg离心2分钟。弃上清，悬浮在1毫升洗涤缓冲液和转让暂停到新的低结合管。在RT孵育5分钟的旋转轮。 </li><li>在在RT 1800 xg离心2分钟，再悬浮于1ml的洗涤缓冲液中离心悬浮液并温育在室温在旋转的轮5分钟。重复4次。对于最后一次洗涤转移到一个新的较低的结合管。 </li><li>除去最大上清液。加7微升的洗涤缓冲液，4倍样缓冲液7微升和3微升10X还原剂至珠，混合和在96℃煮沸5分钟。 </li><li>执行带有int抗体蛋白质印迹分析erest（具体为合作伙伴候选免疫沉淀的蛋白质）。使用输入提取物的1％（5微克），为内源蛋白的水平（ 图3B）的控制。 </li></ol></li></ol></li></ol>

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+motif+N-terminal+to+the+DNA-binding+domains+of+myogenic+bHLH+transcription+factors+mediates+cooperative+DNA+binding+with+pbx-Meis1/Prep1.">A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.</a> Nucleic Acids Res. 27 (18), 3752-3761 (1999).</li><li>Albini, S., Puri, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWI/SNF+complexes,+chromatin+remodeling+and+skeletal+myogenesis:+it's+time+to+exchange!.">SWI/SNF complexes, chromatin remodeling and skeletal myogenesis: it's time to exchange!.</a> Exp Cell Res. 316 (18), 3073-3080 (2010).</li><li>Yahi, H., Fritsch, L., Philipot, O., Guasconi, V., Souidi, M., Robin, P., Polesskaya, A., Losson, R., Harel-Bellan, A., Ait-Si-Ali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Cooperation+between+Heterochromatin+Protein+HP1+Isoforms+and+MyoD+in+Myoblasts.">Differential Cooperation between Heterochromatin Protein HP1 Isoforms and MyoD in Myoblasts.</a> J Biol Chem. 283 (35), 23692-23700 (2008).</li></ol>

The authors have nothing to disclose.

该方法允许详尽的鉴定转录因子的MyoD的合作伙伴。它揭示了MyoD的来诱导分化，即接种HeLa-S3细胞性异源系统MyoD的合作伙伴。因此，根据定义，确定MyoD的合作伙伴普遍表达。这些包括一般和序列特异性转录因子，染色质修饰酶，RNA加工蛋白质激酶（ 表1和2）。自的HeLa-S3细胞对MyoD的诱导转分化抗性，MyoD的活动主要是压制性以及所识别的伙伴可能是MyoD的辅阻遏。这是通过Id蛋白（ 表1和2，图2），已知抑制MyoD的反式激活能力8的存在，突出显示。重要的是，这种压制性的状态对应于成肌细胞增殖的MyoD的状态。事实上，我们证实CBFβ，一个MyoD的那MyoD的相互作用由所描述的方法鉴定的伙伴，可以在增殖C2C12细胞进行检测，但是，当细胞发生分化（ 见图3D）被丢失。然而，要注意的是的呈现方法的主要限制之一是缺乏的MyoD共活化剂的识别是重要的。一致，用已知的MyoD共活化剂，如组蛋白的这种方法无乙酰32进行了鉴定。 MyoD的复合物的纯化或从细胞核或质富集的级分（核小体富集的，NE）（ 图1A）的可溶部分（核盐溶性，SE）提供至少两个主要功能。首先，这种分离增加了，如果MyoD的纯化总核提取物进行的，将被掩盖了非化学计量合作伙伴的代表。其次，这允许的MyoD两个功能亚群的分离:预敷/ EVIC泰德（SE）和染色质结合（NE）。间特异于DNA-未结合的MyoD，许多激酶，转录因子相互作用物，贩卖的蛋白，以及一些染色质remodelers被确定（ 图2）。当充分验证，这样的网络可以提供一个洞察DNA的未结合的MyoD的活性调控的模式。在通过质谱法这两个核区室的MyoD翻译后修饰额外的搜索，能够进一步解开MyoD的活性调控。最后，在一个甘油梯度可溶和染色质结合的MyoD复合物分馏显示，虽然染色质结合的MyoD主要包含在一个复杂的，DNA的未结合的MyoD分布在三个子复合物（ 图1B）。无论是通过MS和/或对蛋白质的候选人免疫印迹这些子复合物的表征应该进一步阐明的MyoD监管的模式。 如上述强调，HeLa细胞不自然expreSS肌肉转录因子MyoD的。它是如此重要的是要确认在骨骼肌模型的发现相互作用（ 图3）。许多报告中的相关模型MyoD的证实和一些在提交TAP标签法鉴定的合作伙伴之间的这种功能的相互作用。这是例如用于抑制素33，DDX17 34，MEIS1 35，CBF 27，HP1 26和SWI / SNF染色质重构复合物36,28,30的情况。 需要注意的是，可以从细胞，例如从成肌细胞，当结合到敏感的MS的低量进行这样的TAP标记纯化。事实上，最近的一篇文章中的成肌细胞30 Flag标记的MyoD的诱导表达描述后MyoD的合作伙伴表征。由于成肌细胞中的稳定，连续的MyoD表达是有害的，另一种方法是加入标志-HA标记成肌细胞内源性的MyoD等位基因通过使用基因组编辑方法，如CRISPR-Cas9 31。值得注意的是，除了所述（多个）标签的可能改变蛋白质的功能和/或关联与结合伴侣，所以该标记（N-或C-末端）的地方，应谨慎选择。在相关的系统中的融合蛋白的功能性测定法必须执行之前TAP标记纯化，以确保标签蛋白是功能性的。内源性蛋白的免疫沉淀避免了这些问题，然而，它依赖于具体和高亲和力的抗体，这是很少可用的可用性。 所描述的方法的另一种增值是纯化蛋白本身和其丰富的伙伴的识别翻译后后修饰的可能性。因此，有此功能的TAP-tag纯化适合于识别，不仅新的交互合作伙伴，而且相关的利息及/或其合作伙伴的新的蛋白质酶的功能。在一个染色质结合蛋白（ 即 ，转录因子，酶）的情况下，此方法因此适于相关的"组蛋白编码"的鉴定。的确，氨基末端组蛋白尾部，这些核小体表面露出，受到多重共价翻译后修饰。组蛋白翻译后修饰赋予所涉及的核小体一个独特的签名。因此，组蛋白N端尾部的修改不同的组合可以改变染色质结构，使基因表达调控。因此，表征关联到一个给定的蛋白能提供深入了解的角色和所研究的染色质结合蛋白22的作用机制这样的修改。 总之，所提出的方法允许的MyoD伙伴的全面鉴定。该TAP-tag纯化提供了其他方法，如GST拉起伏，酵母双杂交和噬菌体展示的替代品。即使对于实际的原因（生产中的Ñ​​大量的核提取物的），我们必须使用异源蜂窝系统中，我们已经能够证实在骨骼肌分化所识别的MyoD伙伴的参与。所获得的数据表明，MyoD的生肌因子似乎与蛋白质从转录调节到RNA结合蛋白的过多相互作用，这表明调节的转录因子的活性的不同的机制。 总之，同样的方法学的方法可以被用于识别的大量的核因子，可能是难以在其特定的细胞背景下研究普遍表达的合作伙伴。

真核多细胞生物是由不同的器官和组织。各功能性组织具有特定的基因图案表达，这是在各个分化步骤中确定。细胞分化涉及特定基因的活化，维持它们的表达，并且通常沉默一组基因，例如那些参与细胞增殖的。骨骼肌分化，或成肌，因而是一个多步骤的过程，与中胚层干细胞的确定为成肌细胞开始，然后导致这些成肌成第一单核，然后多核，肌管的终端分化。因此，成肌细胞被"确定"的细胞，其仍然能够增殖，但他们致力于骨骼肌谱系，因此可以单独使用分化成期间胚胎发育或成年肌肉再生的骨骼肌细胞。骨骼肌终端的过程不同entiation由特定遗传程序，与来自成肌细胞前体细胞的细胞周期，导致的增殖相关的基因，如E2F靶基因1明确沉默永久退出开始协调。实际上，终端分化的过程中，成肌细胞增殖阻滞是之前的骨骼肌特异性基因表达和成肌细胞的融合成肌管2的关键步骤。这样的程序允许成年肌肉干细胞，也称为卫星细胞，在以下骨骼肌损伤再生过程来区分。 哺乳动物肌形成是严重依赖于一个家庭生肌碱性螺旋-环-螺旋（bHLH结构）转录因子的MyoD，Myf5的，MRF4和肌细胞生成素，经常被称为家庭的骨骼肌确定因素或回收设施（肌肉调节因子3）3。他们每个人都在扮演着规范和DIF至关重要的作用骨骼肌细胞的ferentiation和具有特定的表达图案4 5-7 Myf5的和MyoD的活化构成了承诺细胞生肌谱系的决定性步骤，和肌细胞生成素的后续表达触发肌形成与骨骼肌特异基因，如MCK（肌肉肌酸激酶）的活化。生肌的bHLH转录因子与从先前无声轨迹8在肌肉的基因的激活MEF2家族的成员进行合作。它们也刺激骨骼肌基因的转录与普遍存在的bHLH蛋白，E12和E47，已知为E蛋白，其在各个基因调控区8结合所谓的E-盒异二聚体。捻，ID（分化抑制剂）等因素负调节这一过程中，通过与MyoD的对于E蛋白结合8竞争。 MyoD的是引发肌肉终末分化9视为主要参与者</s向上&gt;因为它具有诱导在许多完全分化非肌肉系统10-13一个生肌判定/分化（反分化）方案的能力。事实上，MyoD的强制表达诱导不同的细胞类型，甚至没有从另一个胚胎起源12衍生的转分化。例如，MyoD的可肝细胞，成纤维细胞，黑素细胞，神经母细胞，以及脂肪细胞转化成肌样细胞。的MyoD的反式分化行动涉及的生肌遗传方案的在非肌肉环境中的异常活化（特别是它的靶基因），伴随的原始遗传程序（尤其是增殖基因）的沉默。 在增殖成肌细胞，MyoD的表达，但不能激活其靶基因甚至当它结合到他们的启动子14-16。因此，对于MyoD的在未分化的成肌细胞不断表达的需求仍然相当埃卢西伯。 MyoD的抑制可能因镇压染色质修饰酶的招聘其靶基因在激活染色质重塑酶14,17加载之前增殖成肌细胞。例如，在增殖成肌细胞，MyoD的与转录共阻遏KAP-1，组蛋白脱乙酰酶（HDACs）和压制性赖氨酸甲基转移（KMTs），包括组蛋白H3赖氨酸9或H3K9和H3K27 KMTs相关联，并且积极地抑制其靶基因的表达通过建立本地镇压染色质结构14,17。重要的是，最近的一份报告指出，MyoD的本身由H3K9 KMT G9A从而抑制其反式激活活性16的直接甲基化。 参与非肌肉细胞的MyoD的这种转分化的表观遗传机制在很大程度上是未知的。值得注意的是，一些细胞系是对MyoD的诱导转分化抗性。因此，在HeLa细胞中，MyoD的或者是不活动的，或甚至还不如阻遏，而不是转录激活功能，由于缺乏染色质重塑复杂SWI / SNF 18 BAF60C亚基的表达。因此，这种模式可以选择，以更好地表征的MyoD诱导基因抑制的机制。它也适合于测定的MyoD在其靶位点以诱导压制性染色质环境及其关联伙伴，因此揭开MyoD的依赖性压制性机制在增殖成肌细胞微调终末分化的能力。 在这里，我们通过使用耦合到质谱（MS）表征串联亲和纯化（TAP-标签）描述的MyoD伙伴的标识的协议。使用的HeLa-S3稳定表达的Flag-HA-的MyoD的许可来获得足够的材料来净化从分馏核提取物MyoD的。在异源系统的MyoD伙伴的识别随后validati在一个相关的系统。

<table border="0" cellpadding="0" cellspacing="0" width="543">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Cell lines</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HeLa-S3</td>
			<td style="width:115px;">ATCC</td>
			<td style="width:101px;">CCL-2.2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">C2C12</td>
			<td style="width:115px;">ATCC</td>
			<td style="width:101px;">CRL-1772</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Equipment</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Spinner</td>
			<td style="width:115px;">Corning</td>
			<td style="width:101px;">778531</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Homogenizer :&#160; Dounce homogenizer&#160;</td>
			<td style="width:115px;">Wheaton</td>
			<td style="width:101px;">432-1273 and 432-1271</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Agitating device for spinners</td>
			<td style="width:115px;">Bellco</td>
			<td style="width:101px;">778531</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Sonicator</td>
			<td style="width:115px;">Diagenode</td>
			<td style="width:101px;">UCD 200</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Hemocytometer</td>
			<td style="width:115px;">Marienfeld Superior&#160;</td>
			<td style="width:101px;">640610</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Low-binding tubes</td>
			<td style="width:115px;">Sorenson</td>
			<td style="width:101px;">27210</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Empty spin column</td>
			<td style="width:115px;">Bio-Rad</td>
			<td style="width:101px;">7326204</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Reagents</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">SDS-polyacrylamide 4-12 %&#160; gel</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0336BOX</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">4X loading buffer&#160;</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0007</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10X reducing agent</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0004</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Silver staining kit</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">A8592</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Centrifugal filter units, 10K</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:101px;">UFC501024</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protein G agarose beads</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">P4691</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Protein A/G&#160; Resin</td>
			<td style="width:115px;">Thermo Scientific</td>
			<td style="width:101px;">53132</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Flag resin (anti-Flag M2-agarose affinity gel)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2220</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">HA resin (monoclonal Anti-HA agarose)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2095</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MNase</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">N3755</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Flag free peptide (DYKDDDDK)</td>
			<td style="width:115px;">Ansynth Service BV</td>
			<td style="width:101px;">Custom synthesis</td>
			<td>Resuspend up to 4 mg/mL in 50 mM Tris-HCl, pH 7.8</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">HA free peptide (YPYDVPDYA)</td>
			<td style="width:115px;">Ansynth Service BV</td>
			<td style="width:101px;">Custom synthesis</td>
			<td>Resuspend up to 4 mg/mL in 50 mM Tris-HCl, pH 7.8</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Bicinchoninic acid based protein assay kit : BCA kit</td>
			<td style="width:115px;">Thermo Scientific</td>
			<td style="width:101px;">23225</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Protease inhibitors</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S8830</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:177px;">Luminol-based enhanced chemiluminescence (ECL) HRP substrate</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">34075</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BSA</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9647</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">Sheared salmon sperm DNA (Deoxyribonucleic acid sodium salt from salmon testes)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D1626</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Spermine tetrahydrochloride</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S1141</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Spermidine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S0266</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">G5516</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBS</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D8537</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MOPS running buffer</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM (high glucose)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D0822</td>
			<td>Pre-warm&#160; at 37 &#176;C before use</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Trypsin-EDTA (0.05% phenol red</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;">Serum</td>
			<td>GE healthcare life sciences&#160;</td>
			<td style="width:101px;">PAA A15-102</td>
			<td style="width:151px;">Each lot of serum has to be first tested for your cells.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin and Streptomycin&#160;</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Water (sterile-filtered)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">W3500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Antibodies</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HA from rat (12CA5)</td>
			<td style="width:115px;">Roche</td>
			<td style="width:101px;">11583816001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Flag</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A8592</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MyoD</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>sc-32758</td>
			<td>To use for western blotting</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MyoD</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>SC-760</td>
			<td>To use for immunoprecipitation and western blotting</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">CBFbeta</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">FL-182&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1alpha</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">2HP2G9</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1beta</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">1MOD1A9AS</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1gamma</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">2MOD1GC</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Suv39h1</td>
			<td style="width:115px;">Cell Signaling Technology</td>
			<td style="width:101px;">8729</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">BAF47</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:101px;">612111</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Myf5</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>SC-302</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Tubulin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">T9026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Actin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A5441</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">IgG Mouse</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">SC-2025</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">IgG Rabbit</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">SC-2027</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-rat -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9037</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-rabbit -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A6154&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-mouse -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A4416</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of myod interactome using tandem affinity purification coupled to mass spectrometry

骨骼肌终末分化开始于多能中胚层前体细胞向成肌细胞的承诺。这些细胞具有仍然增殖的能力，或者它们可以分化和融合成多核肌管，其中，还maturate形成肌纤维。骨骼肌终末分化是由多种转录因子的协调动作协调，特别是肌肉调节因子或回收设施的成员（MyoD的，肌细胞生成素，Myf5的，和MRF4），也称为成肌的bHLH转录因子家族。这些因素精心转录调控网络中的染色质重塑复合物合作，以实现骨骼肌肉发生。在这方面，MyoD基因被认为是引发肌肉终末分化的主生肌转录因子。这个概念是由MyoD的与非肌肉细胞转化成骨骼肌细胞的能力增强。在这里，我们描述了用于识别MyoD的一种方法为了阐明参与骨骼肌终端分化的不同因素以详尽的方式蛋白伙伴。长期目的是了解参与骨骼肌基因的调控， 即 ，MyoD的目标的表观遗传机制。 MyoD的伙伴通过从耦合到质谱（MS）表征的异源系统中，随后有关伙伴的骨骼肌终末分化过程中的作用的验证使用串联亲和纯化（TAP-标记）标识。先天性肌无力，肌强直性营养不良，横纹肌肉瘤和缺陷在肌肉再生:生肌因子，或它们的异常调节异常的形式，与许多肌肉障碍相关联。因此，生肌因素提供潜在治疗靶点池肌肉疾病，既涉及导致疾病本身和再生机制，可以提高疾病的治疗机制。因此，详细understan由生肌因素控制分子间相互作用和基因计划的鼎是有效治疗的合理设计是必不可少的。

理解的MyoD活性的调节，我们进行使用生化纯化的MyoD复合物的详尽描述，根据的MyoD的双重标记的形式，随后通过质谱法（MS）的免疫纯化。表达旗HA-MyoD的标记和表达旗HA许可证控制细胞系使用的HeLa-S3细胞，以获得足够的材料通过执行旗HA-MyoD的双亲和纯化，净化MyoD的复杂。 我们分级细胞提取物到细胞质和细胞核分数，然后再分次核分数盐提取（核盐萃取，SE）和丰富的核小体（核小体富集，NE）分数。从这些分离核级分（ 图1，表1和2）的TAP-标签纯化允许解开具有相对低的从头伙伴当在一个特定的亚核车厢本地化undance。此外，这种战略被利用来发现与 DNA结合（NE）的MyoD绑定（SE）的合作伙伴，以获取有关MyoD的活性调控的见解。 对于TAP-tag纯化，使用了标志-HA抗原表位串联系统。小亲水标志和HA抗原决定基与蛋白质功能的干扰最小，并且是抗体 - 抗原相互作用非常方便。抗Flag和抗HA树脂基顺序免疫纯化进行，随后使用标志和HA肽的免疫纯化的复合物的洗脱。洗脱蛋白是然后在SDS-PAGE上运行，以允许所有的蛋白质进入凝胶。凝胶包含所有纯化的蛋白质片断者忌食;将蛋白质提取，胰蛋白酶消化，并通过质谱（MS）来识别。 如图2， </stron从东北部分纯化g&gt;的MyoD的复合物在转录因子和辅抑制高富集。 MS分析揭开了一系列被确认通过这种分析产生的研究，MyoD基因的知名合作伙伴（如程控交换机，身份证，E12 / E47（HTF4），BRG1（SMCA4），MEIS1 ...）和新的合作伙伴（如HP1的， CBF，MBD3，BAF47（SNF5 / INI1）和所有其他的SWI / SNF复合物的亚基...）（ 图2和3）26-28。这揭示了DNA绑定MyoD的合作伙伴以及可能的联合监管，可以建立一个压制性染色质环境光。例如，HP1蛋白，它被确定为所描述的方法MyoD的合作伙伴，是已知结合H3K9甲基化来维持基因镇压和异染色质结构。事实上，HP1抑制导致受损的MyoD靶基因的表达和肌终末分化26 MyoD的转录活性。 进一步分馏甘油梯度SE和网元的MyoD复合物（如在29中所述）揭示在两个亚核区室（ 图1B）的MyoD的子复合物。特别是，SE MyoD的是分布在三个子复合物，而染色质结合的MyoD主要属于一个复杂。 有些TAP标签/ MS透露干扰作用是通过在MyoD的复合物免疫印迹证实。这些包括转录因子的CBF，EBB，MTG8R和SWI / SNF亚基BAF47（SNF5）（ 图3A，左 ）和HP1蛋白（CBX1和CBX3）（ 图3A，右 ）。重要的是，因为HeLa细胞是未肌细胞和不表达的MyoD，有必要确认在成肌细胞（ 图3 BD）新鉴定的相互作用物和MyoD的之间的相互作用。值得注意的是，这样的验证，总的核提取物（不带分离的SE和NE）通常就足够了，whic^ h允许减少用于样品制备成肌细胞的量。 体外相互作用分析为26,27，有利于进一步验证这些结果。最后，这些相互作用的肌肉细胞功能的含义应进一步处理为26-28。 总之，呈现数据显示，普遍表达的MyoD合作伙伴的全球视野和铺平道路，进一步的功能研究的方式更相关的肌肉模型。 <img alt="图1" src="/files/ftp_upload/53924/53924fig1.jpg" /> 图1:MyoD的配合物的串联亲和纯化分离的 （A）的核盐萃取的（SE）或核小体富集的（NE）中分离的双亲和纯化eMyoD复合物的HeLa-S3细胞系稳定核级分的银染。表达旗HA-MyoD基因（MyoD的复合物）的D控制细胞系（模拟）。兆瓦，在千道尔顿分子量标记（kDa的）。箭头指示标志-HA-的MyoD（eMyoD）。这项研究最初发表于37。版权所有美国社会为生物化学与分子生物学。这个数字已经从26修改:故作纯化的车道和eMyoD复杂的SE核部分隔离会立即显示。 （B）的双亲和纯化eMyoD配合物作为（A）中分别于甘油梯度从20％至41％的分级。级分手工收集，浓缩，并通过使用抗Flag抗体免疫印迹（WB）分析。需要注意的几个存在核盐提取（SE）分数次含复合eMyoD- <a href="https://www.jove.com/files/ftp_upload/53924/53924fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" sRC ="/文件/ ftp_upload / 53924 / 53924fig2.jpg"/&gt; 图 2: 染色质结合（核浓缩，NE） 与 核可溶物（核盐溶性，SE）MyoD的合作伙伴 比较 热门 :核盐提取（SE）和核小体富集隔离eMyoD交互者之间的维恩图显示重叠（ NE）部分。核糖体蛋白，翻译起始因子，DNA修复因子和微管蛋白同种型是从分析中排除，因为它们存在于由TAP获得并视为非特异性底各种不同的数据集。在两个SE发现的MyoD的交互件和NE馏分（公共）或特异于级分（唯一的）中的一个是在基团基于其功能注释分割。细胞骨架相关的和其他杂​​项蛋白质未描绘。下划线蛋白是验证要么在HeLa和/或在myoblas MyoD的交互件通过免疫共沉淀TS。 <a href="https://www.jove.com/files/ftp_upload/53924/53924fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53924/53924fig3.jpg" /> 图3:MyoD的交互件的选择集的验证 （A）的MyoD交互器的验证，通过质谱，在HeLa-S3细胞系稳定表达的Flag-HA-的MyoD（eMyoD） 左小组确定:双亲和力Western blot分析核盐提取（SE）或接种HeLa-S3细胞的核小体富集的（NE）馏分稳定表达eMyoD或对照细胞系（模拟），用指定的抗体分离出来 - 纯化MyoD的复合物。 MW，分子量标志物右图:从的HeLa-S3细胞线S的核小体富集级分中分离双亲和纯化的MyoD复合物的Western印迹分析tably表达与指定抗体eMyoD或对照细胞系（模拟）。这个小组最初发表在生物化学杂志。亚希H，弗里奇L，Philipot 0，Guasconi V，Souidi男，罗宾·P，Polesskaya A，Losson R，哈雷尔-Bellan A，艾特-Si系阿里S.生物化学杂志。 2008年8月29; 283（35）:23692-700。 DOI:10.1074 / jbc.M802647200。 EPUB 2008年7月2版权所有美国社会为生物化学与分子生物学。这个数字已经从26修改:警察类型和大小被改变，案文旋转到统一的数字内标签。 MyoD的标为eMyoD以避免与在其他面板呈现内源性IP的混乱。在C2C12成肌细胞鼠MyoD的交互件（BD）验证。 （B）从增殖的C2C12成肌细胞的核总的萃取物用抗BAF47（SNF5）或MyoD的，或用对照IgG产生的抗体用于免疫沉淀（IP）。将所得的沉淀物WB机智分析小时指定抗体。对于抗MyoD的抗体更长（长世博会。）和短（短博览会。）曝光时间显示。输入的萃取物装载到显示内源蛋白的水平。该面板已经发表在28采用知识共享署名（CC BY）许可（http://creativecommons.org/licenses/by/4.0/）。这个数字已经从28修改:警察类型和大小被改变和测试旋转到统一的数字内标签。使用了（C）从增殖的C2C12成肌细胞的核总提取物与针对的MyoD，SUV39H1（阳性对照）产生的抗体免疫沉淀，或对照IgG（阴性对照）。然后将所得的沉淀物进行WB用所示的抗体。这个小组最初发表在生物化学杂志。亚希H，弗里奇L，Philipot 0，Guasconi V，Souidi男，罗宾·P，Polesskaya A，Losson R，哈雷尔-Bellan A，艾特-Si系阿里S.生物化学杂志。 2008年8月29日; 283（35）:23692-700。 DOI:10.1074 / jbc.M802647200。 EPUB 2008年7月2版权所有美国社会为生物化学与分子生物学。这个数字已经从26修改:警察类型和大小被改变，案文旋转到统一的数字内标签。 （D）的核总提取物增殖（prolif）和分化的C2C12成肌细胞（48小时，表示为比较差异。）用于免疫沉淀（IP）与抗MyoD的和Myf5的产生的抗体，或与正常兔IgG和空珠阴性对照。将所得沉淀物WB用所示抗体分析。输入的萃取物装载到显示内源蛋白的水平。该面板已经发表在27采用知识共享署名（CC BY）许可（http://creativecommons.org/licenses/by/4.0/）。这个数字已经从27修改:警察类型和大小被改变，案文旋转到F范围内统一标识igure。与增殖和差异C2C12在二者分离，而不是原来的数字得到的结果的面板。 <a href="https://www.jove.com/files/ftp_upload/53924/53924fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 表1:由MS在从对照细胞系洗脱液鉴定蛋白质通过质谱分析在背景技术蛋白质的减法之后，从核盐萃取的部分分离双亲和纯化eMyoD配合物识别的表 （蛋白质被视为非特异性背景）数据代表四个独立纯化的总和。 <a href="https://www.jove.com/files/ftp_upload/53924/Table_1.xlsx" target="_blank">请点击此处下载此文件。</a> 表2: 蛋白质我的列表从背景蛋白质的减法后的核小体富集的部位分离双亲和纯化eMyoD配合物MS分析dentified。数据代表三个独立纯化的总和。 <a href="https://www.jove.com/files/ftp_upload/53924/Table_2.xlsx" target="_blank">请点击此处下载此文件。</a> 表3.缓冲成分。 <a href="https://www.jove.com/files/ftp_upload/53924/Table_3.xls" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry at JoVE.com

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

identification-myod-interactome-using-tandem-affinity-purification

Nanyang Technological University

Research

JoVE Journal

Biology

9.8K 次观看.  Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité. MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

视频: Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

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

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

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

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

Identification of Protein Interacting Partners Using Tandem Affinity Purification

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

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

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

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

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

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 ...

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

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

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

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

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

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

High-throughput Purification of Affinity-tagged Recombinant Proteins

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

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

高通量的亲和标签的重组蛋白的纯化

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