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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass beads</td>
			<td>BioSpec Products</td>
			<td align="right">11079105</td>
			<td>0.5 mm dia.</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 align="right">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 1:1000</td>
		</tr>
		<tr>
			<td>c-Myc antibody (A-14)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-789</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Purified mouse antibody monoclonal 9E10</td>
			<td>Covance</td>
			<td>MMS-150P</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Ubiquitin (P4G7) monoclonal antibody</td>
			<td>Covance</td>
			<td>MMS-258R</td>
			<td>Primary antibody, dilution 1:1000</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 1:5000</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 1:5000</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 align="right">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

Nanyang Technological University

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

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

科研

生物学

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 ...

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 ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

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.  ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...