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
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?...

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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 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...

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 ...

Research

JoVE Journal

Biology

Measuring Replicative Life Span in the Budding Yeast

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

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

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

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

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

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

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

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

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

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 ...