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>

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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?...

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...

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

Research

JoVE Journal

Biology

12.1K 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, ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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

摘要

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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

高活性酵母核糖体的色谱分离纯化