Name: identification of protein complexes with quantitative proteomics in s. cerevisiae
Uploaded: 2009-03-04T00:00:00
Description: Watch this Scientific Journal Video about Identification of protein complexes with quantitative proteomics in S. cerevisiae at JoVE.com

材料和方法<ol><li>酵母菌株<ul><li>在这项研究中使用的所有菌株的基础上BY4742背景。 SILAC控制应变（ 垫的，HIS3，LEU2，URA3，lys2和arg4：G418）是由交配arg4缺失株（ 垫，HIS3，URA3，LEU2，和arg4：G418）BY4742（垫阿尔法， HIS3，LEU2，URA3和lys2），减数分裂单倍体获得四分体夹层。因此，控制应变是赖氨酸和精氨酸营养缺陷型。使用矢量pBS1479 PCR介导的同源重组诱饵应变。因此，在这项?...

<ol>
	<li>Loewen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+cortical+ER+in+yeast+is+required+for+normal+septin+organization.">Inheritance of cortical ER in yeast is required for normal septin organization.</a> Jour. Cell Biol. 179, 467-483 .</li><li>Ong, S., Foster, L. J., Hoog, C. L., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric-based+approaches+in+quantitative+proteomics.">Mass spectrometric-based approaches in quantitative proteomics.</a> Methods. 29, 124-130 .</li><li>Puig, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tandem+Affinity+Purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The Tandem Affinity Purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 .</li><li>Amberg, D. C., Burke, D. J., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Yeast+Genetics.">Methods in Yeast Genetics.</a> , 123-125 .</li></ol>

在纯化过程中保存的等分应包括：（1）事先批准的细胞裂解液，（2）约束的一小部分，（3）不作承诺的一小部分，以及（4）洗脱组分。我们建议由西方上述等份分析的蛋白质含量，杂交使用抗TAP抗体或银染色，以反映实验的约束力和洗脱效率。一个银染凝胶和印迹的例子，如图1所示。 由于SILAC我们提供公正和量化的测量与蛋白结合的合作伙伴，我们只能做的TAP纯化的第一?...

identification of protein complexes with quantitative proteomics in s. cerevisiae

血脂，细胞膜功能的细胞过程中条块分割的壁垒和积木，最近，作为重要的细胞内信号分子。然而，与蛋白质，脂类是小的疏水性分子的交通主要是由不良描述nonvesicular路线，这是假设发生在膜接触点（MCSS）。热对流是内质网（ER）与一个合作伙伴的细胞器，如细胞膜（下午）直接的身体接触的地区。丰富脂质合成酶的ER - PM的热对流的ER部分，这表明，脂质合成是针对这些网站和暗示的MCS是重要的脂质交通。酵母是一种理想的模型来研究ER - PM的，因为它的丰富的MCS与超过1000个联系人，每个细胞中，和他们在所有真核生物中保守性，。发现的蛋白质构成的MCS是至关重要的理解血脂交通是如何在细胞内完成，以及它们如何作为信号分子的行为。我们发现，所谓的Scs2p一个ER本地化ER - PM的MCS，其形成的重要。我们专注于揭露Scs2p分子伙伴。鉴定蛋白质复合物的传统依赖于首​​先解决凝胶电泳凝胶的蛋白条带的消化和分析肽质谱，纯化的蛋白质样品。这往往限制了研究的蛋白质的一小部分。此外，蛋白质复合物的暴露变性或在手术过程中的非生理条件。为了规避这些问题，我们已实施了大规模的定量蛋白质组学技术提取公正和量化的数据。我们用稳定同位素标记氨基酸在细胞培养（SILAC）纳入蛋白质在无标记的控制应变主食同位素原子核。标签的文化和无标记的，SILAC标记的文化等量混合在一起，并在液氮研磨溶解。然后，我们开展的亲和纯化过程拉下蛋白质复合物。最后，我们沉淀的蛋白质样品，这是准备通过高性能的液相色谱/串联质谱分析。最重要的是，在控制菌株的蛋白质是由重同位素标记会产生一个质量/充电的转变，可对未标注蛋白质量化诱饵应变。因此，污染物，或者非特异性结合，可以很容易地消除。通过使用这种方法，我们已经确定了一些新的蛋白质，定位于ER - PM的MCS。在这里，我们提出了我们的方法的详细描述。

Watch this Scientific Journal Video about Identification of protein complexes with quantitative proteomics in S. cerevisiae at JoVE.com

Identification of protein complexes with quantitative proteomics in S. cerevisiae

在这里，我们描述了一个新的量化确定在酿酒酵母中的蛋白质复合物的蛋白质组学技术。在这项研究中，我们一起串联质谱法，具有高特异性识别ER蛋白，Scs2p的具有约束力的合作伙伴的亲和纯化使用的SILAC方法。

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Research

JoVE Journal

Biology

Assay for Adhesion and Agar Invasion in S. cerevisiae

化验粘附和酿酒酵母琼脂入侵

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

科研

生物学

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Identification of Post-translational Modifications of Plant Protein Complexes

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...