Name: protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80
Uploaded: 2015-05-03T14:00:00.000+00:00
Duration: 12 min 28 s
Description: Watch this Scientific Journal Video about Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 at JoVE.com

We thank Dr. Oliver Kerscher for support and advice and members of the Basrai laboratory for their support and comments on the paper. This work was supported by the National Institutes of Health Intramural Research Program.

1.生长酵母细胞的<ol><li>在一个小烧瓶中接种于30ml YPD（ 表1）的酵母细胞。孵育在30℃过夜振荡培养。 </li><li>在50 YPD毫升稀释细胞至0.2的光密度在600nm（OD 600 = 0.2）和孵育在30℃下振荡。 </li><li>生长培养物以1.0在600nm（OD 600 = 1.0）的光密度。 </li><li>离心细胞5分钟，在2000×g离心并弃去上清液。 </li><li>重悬在40毫升无菌水中，并离心将细胞沉淀5分钟，在2000×g离心以洗涤细胞。弃去上清液并储存细胞沉淀在-20℃下。 </li></ol> 2.提取的蛋白质<ol><li>使用Ni-NTA超流珠或悬浮细胞在0.5ml冰冷胍缓冲液（表1）为下拉测定法缓冲液A（ 表1）进行免疫沉淀。保持在冰上管在任何时候。 </li><li>转移至2μm升螺丝帽筒。 </li><li>加入的玻璃珠相同体积（表材料 ）。 </li><li>珠击败细胞在微型珠打浆机（表材料）在室温下2分钟，然后放置在冰上2-3分钟。重复此三次。 </li><li>涡旋高速在4℃下30-60分钟。检查细胞可视化在显微镜下，以确保细胞溶解。 注:裂解的细胞会出现暗鬼，缺乏边界或定义形状。最适的至少80％的细胞应被裂解。 </li><li>穿刺在使用推销和地点管底部的孔中收集的15毫升锥形管中（螺丝帽要宽松）。 </li><li>离心在1000×g离心1分钟，以收集裂解液。 </li><li>将裂解物转移到微量离心管中。 </li><li>离心机以15,000×g离心30分钟，在4℃下以收集所提取的蛋白质。 </li><li>使用蛋白阿萨测量所提取的蛋白质的浓度ÿ试剂盒（表材料 ），并归一所有的提取物，以含有蛋白质的量相同。使总体积为1毫升，合适的缓冲液。 注:从50 OD 600细胞获得约5毫克总蛋白。 </li><li>保存50微升（250微克蛋白质，如果从步骤2.10中提取的总蛋白为5毫克），全细胞提取物（WCE）。加入50微升2×Laemmli样品缓冲液（ 表1），并在一个热块孵育在100℃下进行3-5分钟。负载10微升进行Western印迹分析（第5节:Western blot分析）一SDS-PAGE凝胶每个样品。 </li></ol> 3.净化HF-SMT3偶联物<ol><li>得到的Ni-NTA超流珠（表材料 ）所需的实验（100微升每个样品珠）通过低速离心（800-1500×g离心），持续1分钟。去除上清。 </li><li>洗珠五倍用1毫升的PBS（表材料）中:反转顶过底部，直到珠悬浮，收集珠低速离心，去除上清。 </li><li>暂停珠在1ml胍缓冲液（ 表1），并分装成对应于样本被处理管的数量。收集珠低速离心，去除上清。通过反向顶底以上的混合珠好。 注:通过反向顶底多珠混合很好。 </li><li>至100微升的Ni-NTA超流珠:对于每个样品，（蛋白质提取步骤2.11）加950微升其余WCE的。 </li><li>孵育在摇摆平台上在4℃至少4小时或过夜。 </li><li>离心机在800-1500×g离心在4℃下1分钟。 </li><li>节省50微升的上清液（SUP）。加入50微升2×Laemmli样品缓冲液并在一个热块孵育在100℃下进行3-5分钟。负载10微升进行Western印迹分析（第5节的SDS-PAGE凝胶每个样品:Western印迹肛门ysis）。 </li><li>洗涤珠子一次用1ml胍缓冲5-10分钟在摇摆平台上。 </li><li>洗涤珠粒5倍用1ml 5-10分钟破碎缓冲液（ 见表1）在摇摆平台上的。 </li><li>重悬珠子在90微升2×Laemmli样品缓冲液中。 </li><li>加10微升1M咪唑。 注意:咪唑有助于解离从的Ni-NTA珠的带有His标记的蛋白质，由于咪唑和珠粒间竞争相互作用。 </li><li>在3-5分钟的热块孵育100℃。 </li><li>旋涡，然后离心13000 XG 30秒。将上清液转移到新的管中。 </li><li>负荷10-20微升的SDS-PAGE凝胶进行Western印迹分析每个样品的（第5节:Western blot分析）。 注意:10-20微升相当于0.5-1.0毫克输入的，如果5毫克WCE的被使用。 </li></ol> 4.免疫沉淀Myc标签动粒蛋白<ol><li>获得抗c-Myc的琼脂糖亲和凝胶的tibody（表材料 ）所需的实验（25微升每个样品树脂）通过低速离心（800-1500×g离心）。去除上清。 </li><li>用1毫升缓冲液A洗涤树脂5倍:反转顶过底，直到树脂再悬浮，收集树脂低速离心，去除上清。 </li><li>暂停树脂在1ml缓冲液A和等分成对应于样本被处理管的数量。收集树脂低速离心，去除上清。 注:通过反向顶过底混合树脂很好。 </li><li>添加950微升剩余WCE（来自步骤2.11:蛋白质的提取），以25微升树脂。 </li><li>孵育在摇摆平台上在4℃下过夜。 </li><li>离心机在800-1500×g离心在4℃下1分钟。 </li><li>节省50微升的上清液（SUP）。加入50微升2×Laemmli样品缓冲液并在一个热块孵育在100℃下进行3-5分钟。罗广告10微升进行Western印迹分析的SDS-PAGE胶（第5节:Western blot分析），每个样品。 </li><li>用1毫升缓冲液A洗涤树脂5倍:反转顶过底，直到树脂再悬浮，收集树脂低速离心，去除上清。 </li><li>重悬在100微升SUMEB样品缓冲液（ 见表1）的树脂。 注意:SUMEB样品缓冲液含有8M尿素和1％SDS（强变性条件）。 </li><li>在3-5分钟的热块孵育100℃。 </li><li>旋涡，然后离心13000 XG 30秒。将上清液转移到新的管中。 </li><li>负荷10-20微升的SDS-PAGE凝胶进行Western印迹分析每个样品的（第5节:Western blot分析）。 注意:10-20微升相当于0.5-1.0毫克输入的，如果5毫克WCE的被使用。 </li></ol> 5. Western印迹分析<ol><li>加载蛋白提取物在4-12％Bis-Tris凝胶（表材料）和性能的ORM电泳在120伏90分钟（电泳缓冲液:表材料 ）。 </li><li>从凝胶转移蛋白用传送设备硝酸纤维素膜（表材料 ）在30 V 90分钟（传输缓冲区:表材料 ）。 </li><li>方框中的5％乳/ 1×TBST（ 表1）的膜1小时，在室温下进行。 </li><li>孵育膜中的5％乳/ 1×TBST过夜，在4℃下一级抗体（表材料 ）。为初级抗体，可使用的稀释度1:1000（抗Flag和抗泛素抗体）或1:5000（抗Myc抗体）。 </li><li>每10分钟用1×TBST洗膜3次。 </li><li>孵育膜与二级抗体（表材料 ）中的5％乳/ 1×TBST 1小时，在室温下进行。使用1:5000稀释的二抗的。 </li><li>每10分钟用1×TBST洗膜3次。 </li><li>孵育膜ECL worki纳克溶液（表材料 ）5分钟。 </li><li>暴露的膜感蓝X射线胶片（表材料 ）和使用自动显影剂（表材料 ）开发。 </li></ol>

<ol>
	<li>Johnson, P. R., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-1:+Ubiquitin+gains+weight.">SUMO-1: Ubiquitin gains weight.</a> Trends Cell Biol. 7, 408-413 (1997).</li><li>Hewawasam, G., 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=Psh1+is+an+E3+ubiquitin+ligase+that+targets+the+centromeric+histone+variant+Cse4.">Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4.</a> Mol Cell. 40, 444-454 (2010).</li><li>Ranjitkar, P., 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=An+E3+ubiquitin+ligase+prevents+ectopic+localization+of+the+centromeric+histone+H3+variant+via+the+centromere+targeting+domain.">An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain.</a> Mol Cell. 40, 455-464 (2010).</li><li>Au, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+of+the+N+terminus+of+budding+yeast+histone+H3+variant+Cse4+in+ubiquitin-mediated+proteolysis.">A novel role of the N terminus of budding yeast histone H3 variant Cse4 in ubiquitin-mediated proteolysis.</a> Genetics. 194, 513-518 (2013).</li><li>Akiyoshi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mub1/Ubr2+ubiquitin+ligase+complex+regulates+the+conserved+Dsn1+kinetochore+protein.">The Mub1/Ubr2 ubiquitin ligase complex regulates the conserved Dsn1 kinetochore protein.</a> PLoS Genet. 9, e1003216 (2013).</li><li>Biggins, 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=The+conserved+protein+kinase+Ipl1+regulates+microtubule+binding+to+kinetochores+in+budding+yeast.">The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast.</a> Genes Dev. 13, 532-544 (1999).</li><li>Cheeseman, I. M., 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=Phospho-regulation+of+kinetochore-microtubule+attachments+by+the+Aurora+kinase+Ipl1p.">Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p.</a> Cell. 111, 163-172 (2002).</li><li>Liu, D., Lampson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+kinetochore-microtubule+attachments+by+Aurora+B+kinase.">Regulation of kinetochore-microtubule attachments by Aurora B kinase.</a> Biochem Soc Trans. 37, 976-980 (2009).</li><li>Montpetit, B., Hazbun, T. R., Fields, S., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sumoylation+of+the+budding+yeast+kinetochore+protein+Ndc10+is+required+for+Ndc10+spindle+localization+and+regulation+of+anaphase+spindle+elongation.">Sumoylation of the budding yeast kinetochore protein Ndc10 is required for Ndc10 spindle localization and regulation of anaphase spindle elongation.</a> J Cell Biol. 174, 653-663 (2006).</li><li>Furth, N., 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=Exposure+of+bipartite+hydrophobic+signal+triggers+nuclear+quality+control+of+Ndc10+at+the+endoplasmic+reticulum/nuclear+envelope.">Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.</a> Mol Biol Cell. 22, 4726-4739 (2011).</li><li>Li, L., 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=Anaphase-promoting+complex/cyclosome+controls+HEC1+stability.">Anaphase-promoting complex/cyclosome controls HEC1 stability.</a> Cell Prolif. 44, 1-9 (2011).</li><li>Takahashi, Y., Yong-Gonzalez, V., Kikuchi, Y., Strunnikov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIZ1/SIZ2+control+of+chromosome+transmission+fidelity+is+mediated+by+the+sumoylation+of+topoisomerase+II.">SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II.</a> Genetics. 172, 783-794 (2006).</li><li>Liu, C., Apodaca, J., Davis, L. E., Rao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+inhibition+in+wild-type+yeast+Saccharomyces+cerevisiae+cells.">Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells.</a> Biotechniques. 42, 158 (2007).</li><li>Kitamura, E., Tanaka, K., Kitamura, Y., Tanaka, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetochore+microtubule+interaction+during+S+phase+in+Saccharomyces+cerevisiae.">Kinetochore microtubule interaction during S phase in Saccharomyces cerevisiae.</a> Genes Dev. 21, 3319-3330 (2007).</li><li>Gascoigne, K. E., Cheeseman, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetochore+assembly:+if+you+build+it,+they+will+come.">Kinetochore assembly: if you build it, they will come.</a> Curr Opin Cell Biol. 23, 102-108 (2011).</li></ol>

The authors have nothing to disclose.

表位标记，如HA，Myc基因，旗，和GST被广泛用于蛋白质的生化分析。菌株与HF-SMT3和Myc标签动粒蛋白质，如Ndc10和Ndc80的构造，有利于翻译后修饰如SUMO化和泛素化的检测。 HF-SMT3 pull down实验允许SUMO化着丝粒蛋白，Ndc10和Ndc80（ 图1）的检测。亲和纯化方案和Western印迹分析使用抗Flag抗体建立的HF-SMT3和目标蛋白质之间相互作用的特异性，因为没有在无HF-SMT3对照株检测修饰的蛋白质。使用对蛋白质底物的Myc的标签的验证SUMO化Ndc10和Ndc80的在HF-SMT3缀合物的存在。此外，既Ndc10和Ndc80的蛋白修饰减少在诺考达唑处理的细胞（ 图1C和1D）。降低Ndc10的SUMO化修饰响应诺考达唑treatmen吨，但不Ndc80，先前报道Montpetit 等人 9。这可能是由于在实验方案和使用定制的抗的SUMO抗体的差异。我们的研究结果为动粒基板SUMO化表明，一个类似的协议可以用来研究其它候选SUMO衬底。 为了检测泛素化，Myc标签动粒蛋白第一免疫沉淀和IP级分用抗泛素抗体（ 图2）。 Ndc10和Ndc80的梯状图案增加当细胞以MG132处理以抑制蛋白酶体功能（ 图2B），这表明这些蛋白的蛋白酶体的底物。知识产权馏分未能检测Ndc10或Ndc80的蛋白修饰在蛋白质印迹分析用抗Flag或抗SMT3抗体（数据未示出）。这可能是由于技术原因，例如SUMO化衬底在IP级分或干涉的级别E从表位标签。 IP协议的进一步优化，例如盐和Western印迹条件的浓度应促进感兴趣的任何蛋白质的多个翻译后修饰的检测。目前，SUMO化，最好由HF-SMT3检测pull down实验接着候选SUMO基板的蛋白质印迹（ 图1）。 若干细节影响蛋白质纯化效率和检测翻译后修饰的能力。首先，所有管应保持在冰上，以抑制非指定的蛋白酶。第二，以1:1的比例的细胞悬液以玻璃珠是至关重要的珠打浆机和/或涡旋以破碎细胞。细胞裂解应该通过检查显微镜下的细胞进行监测。第三，这是非常重要的，以制备澄清的裂解物用于下拉测定法或IP。短或低速离心可以向在裂解物的细胞碎片的污染，这影响到DETE的能力克拉的翻译后修饰。最后，珠子在1.5 ml的微量离心管中的大量澄清的裂解（约1毫升）的剧烈摇晃确保珠完全暂停下拉法或IP地址。总之，如这里描述的蛋白质纯化和检测方法提供重要的机械见解着丝粒蛋白质的PTM如何影响它们的结构和组件，用于忠实染色体分离14，15。

泛素化和SUMO化允许泛素和小泛素样修饰的缀合（SUMO; SMT3在酿酒酵母 1）到目标蛋白。着丝粒蛋白的翻译后修饰影响其细胞水平和蛋白质 - 蛋白质相互作用中不同的细胞周期阶段，以确保忠实染色体分离。例如，蜂窝Cse4 / CENP-A和外着丝粒蛋白DSN1的水平通过泛素介导的蛋白水解以确保基因组稳定性2-5调节。不正确动粒微管附件失稳要求IPL1 /极光酶b激酶，磷酸化而与Dam1复合Ndc80直接与微管​​6-8交互。尽管年过七旬动粒蛋白的鉴定，也有极少数的研究，调查这些蛋白质翻译后修饰用， 例如 ，泛素和SUMO的修改。一个主要的限制是为了保护PTM的能力s纯化和定制抗体的缺乏检测翻译后修饰如蛋白修饰，磷酸化，甲基化，以及其他的期间。 SUMO化着丝粒蛋白的表征Ndc10，Cep3，Bir1和Ndc80利用自定义抗体9。此外，Ndc10已经牵涉作为底物泛素化10。人类Hec1（Ndc80在酿酒酵母 ）也底泛素化，通过APC / C-hCdh1 E3连接酶调节11。因此，Ndc10和Ndc80是很好的候选方案的最优化同时检测SUMO化和泛素化的S.酵母 。 为了方便SUMO化的标识，我们构建了表达HF-SMT3和Myc标签Ndc10或Ndc80株。使用表位标签（HF:的His6-标志）的背景最小化由于交叉反应性是在多克隆血清了针对一个候选蛋白质经常观察到。我们设计了一个协议，以亲和纯化HF的SMT3偶联物，然后用商业抗Flag和抗Myc抗体检测sumolyated Ndc10和Ndc80在纯化SMT3制剂的存在。对于泛素化，我们设计了一个修饰免疫协议，保留了Myc基因标记着丝粒蛋白的泛素化和进行Western blot分析与商业的抗泛素的抗体来检测Ndc10和Ndc80的泛素化。

<table><tbody><tr><td>Glass beads</td><td>BioSpec Products</td><td>11079105</td><td>0.5 mm diameter</td></tr><tr><td>Mini beadbeater</td><td>BioSpec Products</td><td>#693</td><td>8 cell disrupter</td></tr><tr><td>Ni-NTA superflow</td><td>Qiagen</td><td>30430</td><td>100 ml</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8215</td><td>1 ml</td></tr><tr><td>Anti-c-Myc agarose affinity gel antbody produced in rabbit</td><td>Sigma-Aldrich</td><td>A7470</td><td>1 ml</td></tr><tr><td>Monoclonal anti-Flag M2 antibody produced in mouse</td><td>Sigma-Aldrich</td><td>F1804</td><td>Primary antibody, dilution 1:1,000</td></tr><tr><td>c-Myc antibody (A-14)</td><td>Santa Cruz Biotechnology</td><td>sc-789</td><td>Primary antibody, dilution 1:5,000</td></tr><tr><td>Purified mouse antibody monoclonal 9E10</td><td>Covance</td><td>MMS-150P</td><td>Primary antibody, dilution 1:5,000</td></tr><tr><td>Ubiquitin (P4G7) monoclonal antibody</td><td>Covance</td><td>MMS-258R</td><td>Primary antibody, dilution 1:1,000</td></tr><tr><td>ECL Rabbit IgG, HRP-linked whole Ab</td><td>GE Healthcare Life Sciences</td><td>NA934V</td><td>Secondary antibody, dilution 1:5,000</td></tr><tr><td>ECL Mouse IgG, HRP-Linked Whole Ab</td><td>GE Healthcare Life Sciences</td><td>NA931V</td><td>Secondary antibody, dilution 1:5,000</td></tr><tr><td>DC protein assay</td><td>Bio-Rad</td><td>500-0116</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Novex</td><td>LC2001</td><td>0.45 mm pore size</td></tr><tr><td>NuPAGE 4-12% Bis-Tris Protein Gels</td><td>Novex</td><td>NP0321BOX</td><td>1.0 mm, 10 well</td></tr><tr><td>NuPAGE MES SDS Running Buffer</td><td>Novex</td><td>NP0002</td><td>20x</td></tr><tr><td>NuPAGE Transfer Buffer</td><td>Novex</td><td>NP0006-1</td><td>20x</td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Thermo Scientific</td><td>34078</td><td></td></tr><tr><td>10x PBS pH7.4</td><td>GIBCO</td><td>70011-044</td><td></td></tr><tr><td>Blue sensitive X-Ray film</td><td>Dbio</td><td>DBOF30003</td><td></td></tr><tr><td>Automatic developer</td><td>Kodak</td><td>M35AX-OMAT</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>M1404</td><td>50 mg</td></tr><tr><td>MG-132</td><td>Selleck Chemicals</td><td>S2619</td><td>25 mg</td></tr></tbody></table>

protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80

翻译后修饰（翻译后修饰），例如磷酸化，甲基化，乙酰化，泛素化，和蛋白修饰，调节许多蛋白质​​的细胞功能。与着丝粒DNA着丝粒关联的蛋白质翻译后修饰调解忠实染色体分离，以维持基因组稳定性。生物化学方法如质谱和Western blot分析最常用于识别翻译后修饰的。这里，一个蛋白质纯化方法进行说明，该技术同时SUMO化的动粒蛋白，Ndc10和Ndc80，在酿酒酵母和泛素化的检测。表达组氨酸-Flag标签SMT3（HF-SMT3）和Myc标签Ndc10或Ndc80构建并用于我们的研究的菌株。对于检测蛋白修饰，我们设计了一个协议，以亲和通过使用镍珠纯化His标签的SUMO化蛋白质并用于Western印迹分析用抗Myc抗体检测SUMO化Ndc10一个第二Ndc80。为了检测泛素化，我们设计了一个协议的Myc标签蛋白免疫沉淀和使用免疫印迹分析与抗泛素的抗体，表明Ndc10和Ndc80被泛素化。我们的结果表明，在带有His标记感兴趣的表位标记的蛋白标记的SMT3应变便于多个翻译后修饰的检测。未来的研究应该允许开发这种技术的鉴定和表征蛋白质的相互作用是依赖于特定的PTM。

以检测的着丝粒蛋白质Ndc80和Ndc10蛋白修饰，菌株与HF-SMT3和Myc标签着丝粒蛋白（Ndc80或Ndc10）构建（ 表2），如前所述9,12。 HF-SMT3缀合物使用Ni-NTA珠的亲和纯化。纯化HF的SMT3的Western印迹分析用抗Flag抗体允许检测SUMO底物是不存在于无HF-SMT3（ 图1A和1B，左图）对照菌株的SUMO化形式。正如预期的，多种形式的SUMO基板在HF-SMT3应变进行检测。我们接下来确定是否Ndc80和Ndc10存在于纯化的HF-SMT3缀合物通过执行使用抗Myc抗体免疫印迹分析。多个频带那名的分子量高于该Ndc80或Ndc10被清楚地检测到（ 图1A和1B，右图）。此外，无论是Ndc80和Ndc10的多频段显着降低，诺考达唑处理的细胞（ 图1C和1D）。这些结果表明，蛋白质纯化和蛋白质印迹分析在此描述允许Ndc80和Ndc10的SUMO化的检测中，如先前所描述9。 为了检测Ndc80和Ndc10，泛素化Myc标签Ndc80或Ndc10进行免疫沉淀（IP）和用抗Myc及抗泛素抗体（ 图2）进行免疫印迹分析。的全细胞提取物（WCE）和上清液（SUP）分析证实Ndc80-Myc和Ndc10-Myc的（ 图2A）的表达。在WCE的低分子量条带可能代表降解产物。用抗Myc的IP样品显示多种高分子量条带，这表明Ndc80和Ndc10含有翻译后修饰。用抗泛素抗体的IP样品的梯状图案表明Ndc80和Ndc10被泛素化（ 图2A，α-UB）。 Ndc80和Ndc10的梯状图案通过与蛋白酶体抑制剂（MG132）（ 图2B，α-UB）处理，得到提高，进一步证实了这些带代表聚ubiqutination。代表结果表明，无论Ndc80和Ndc10是SUMO化修饰和泛素化的S.基板酵母和所描述的蛋白质纯化方法可用于检测翻译后修饰如SUMO化和泛素化是有用的。据我们所知，这是首次报道Ndc80在S.酵母是一种底物泛素化，虽然人类同源Hec1的泛素介导的蛋白质水解，已经出版11。 <img alt="图1" src="/files/ftp_upload/52482/52482fig1.jpg" /> 图1:sumolyated基板的纯化允许的着丝粒蛋白Ndc80和Ndc10鉴定为SUMO基板镨。oteins进行提取，制备和Western blot分析分析HF-SMT3结合物SDS-PAGE分离后。 （A和B）Ndc80和Ndc10的SUMO化。蛋白质分别提取对数生长期细胞YPD。左面板:分别检测用抗Flag抗体共有的SUMO衬底。右板:SUMO化Ndc80或Ndc10物在HF-SMT3偶联物检测时用抗Myc抗体探测。 Ndc80和Ndc10的（C和D）SUMO化被降低诺考达唑处理的细胞。 （ - ）对数生长的细胞在YPD分别用DMSO或DMSO + 20微克/毫升诺考达唑（+），2小时，在30℃。 HF-SMT3结合物经Western blot分析使用抗Myc抗体进行分析。使用同基因的酵母菌株是（A和C）YPH1800（Ndc80-Myc的），YMB7862（他-旗SMT3 Ndc80-Myc的）和（B和D）YPH1734（Ndc10-Myc的），YMB7867（他-FLAG-SMT3 Ndc10-Myc蛋白）。 <img alt="图2" src="/files/ftp_upload/52482/52482fig2.jpg" /> 图2:的着丝粒蛋白质Ndc80和蛋白质和免疫沉淀的Ndc10提取泛素化被作为在协议中所述进行。 （A）Ndc80和Ndc10被泛素化。蛋白质分别提取对数生长期细胞YPD。全细胞提取物（WCE），上清液（SUP）和免疫沉淀（IP）的级分进行SDS-PAGE。蛋白质印迹法检测的Myc标记和泛素化蛋白质与抗Myc及抗泛素抗体，分别。 Ndc80和Ndc10（B）的泛素化被增强与蛋白酶体抑制剂MG132处理的细胞。对数生长的细胞在YPD治疗用DMSO（ - ）或DMSO +50μM的MG132（+），在SDS的0.003％，在30℃的存在下进行3小时，如前所述13 </sup&gt;。使用抗Myc或抗泛素抗体IP分数蛋白质印迹进行。使用同基因酵母菌株YMB7278（他-旗SMT3），YMB7862（他-旗SMT3 Ndc80-Myc蛋白）和YMB7867（他-旗SMT3 Ndc10-Myc蛋白）。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 解 </td><td> 组件 </td></tr><tr><td> YPD </td><td> 1％酵母提取物，2％细菌用蛋白胨，2％葡萄糖</td></tr><tr><td>缓冲胍</td><td>的0.1M的Tris-HCl pH 8.0的，6M的盐酸胍，0.5M NaCl的</td></tr><tr><td>缓冲液A </td><td>的50mM的Tris-HCl pH为7.5，50 mM氯化钠，0.2％的Triton X-100，1×蛋白酶抑制剂</td></tr><tr><td>破碎缓冲液</td><td>的0.1M的Tris-HCl pH 8.0的，20％甘油，1mM PMSF </td></tr><tr><td> SUMEB样品缓冲液</td><td> 1％SDS，8M尿素，10mM的MOPS pH 6.8的10毫摩尔EDTA，0.01％溴酚蓝</td></tr><tr><td> 2X Laemmli样品缓冲液</td><td> 100毫米的Tris-HClpH为6.8，4％SDS，20％甘油，0.2％溴酚蓝（使用前:添加β-巯基乙醇（BME），以200毫米的最终浓度） </td></tr><tr><td> 1X TBST </td><td> 137毫氯化钠，20毫摩尔Tris-HCl pH为7.5，0.1％Tween-20的</td></tr></tbody></table> 表1:解决方案。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>在这项研究中使用*表2酵母菌株。 </td><td></td><td></td><td></td></tr><tr><td>株</td><td>亲</td><td>基因型</td><td>参考</td></tr><tr><td> BY4741 </td><td></td><td>的MATahis3Δ1leu2Δ0met15Δ0ura3Δ0 </td><td>开放生物</td></tr><tr><td> BY4742 </td><td></td><td>的MATahis3Δ1leu2Δ0lys2Δ0ura3Δ0 </td><td>开放生物</td></tr><tr><td> YMB7278 </td><td> BY4741 / BY4742 </td><td>的MATahis3Δ1leu2Δ0lys2Δ0乌尔a3Δ0HF-SMT3 :: LEU2 </td><td>这项研究</td></tr><tr><td> YPH1734 </td><td></td><td>的MATa ura3-52 lys2-801 ade2-101his3Δ200leu2Δ1trp1Δ63NDC10-13Myc :: kanMX6 </td><td> Montpetit 等人 ，2006年</td></tr><tr><td> YPH1800 </td><td></td><td>的MATa ura3-52 lys2-801 ade2-101his3Δ200leu2Δ1trp1Δ63NDC80-13Myc :: His3MX6 </td><td> Montpetit 等人，2006年</td></tr><tr><td> YMB7862 </td><td> YMB7278 / YPH1800 </td><td> HIS3的MATa URA3 LEU2 TRP1 ADE2 HF-SMT3 :: LEU2 NDC80-Myc蛋白:: His3MX6 </td><td>这项研究</td></tr><tr><td> YMB7867 </td><td> YMB7278 / YPH1734 </td><td> HIS3的MATa URA3 LEU2 TRP1 LYS2 HF-SMT3 :: LEU2 NDC10-Myc蛋白:: kanMX6 </td><td>这项研究</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> *所有的酵母菌株都来源于酿酒酵母S288C。 </td><td></td><td></td><td></td></tr></tbody></table><p类="jove_content"&gt; 表2:本研究中所用的酵母菌株。

Watch this Scientific Journal Video about Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 at JoVE.com

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

此稿件描述了SUMO化修饰和着丝粒蛋白，Ndc10和Ndc80的泛素化的检测，在芽殖酵母 。

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

protein-purification-technique-that-allows-detection-sumoylation

Universidad San Sebastian

Research

JoVE Journal

Biology

12.0K 次观看.  National Cancer Institute, National Institute of Health. 此稿件描述了SUMO化修饰和着丝粒蛋白，Ndc10和Ndc80的泛素化的检测，在芽殖酵母 。 

视频: Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

科研

生物化学

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

生物学

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

E3泛素连接酶在哺乳动物细胞裂解物中底物泛素化的评价

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

从哺乳动物细胞中纯化泛素化p53蛋白

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

测量泛素化、SUMO 化和 ADP 核糖基化 DNA-蛋白质交联

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

作者聚焦: DNA 蛋白交联及其翻译后修饰的定量检测  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

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此视频中的章节

通过肽浓缩和质谱检测蛋白质泛化位点检测

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

蛋白泛素化的检测

E3泛素连接酶在哺乳动物细胞裂解物中底物泛素化的评价

从哺乳动物细胞中纯化泛素化p53蛋白

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰