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，在这项研究中所使用的抗原表位标记的串联亲和纯化（TAP），标签（3）。 </li></ul></li><li> SILAC： <ul><li> SILAC控制株生长在最小辍学培养基，98％的L -赖氨酸- 4 ,4,5,5 - D4（0.03 g / L的，剑桥同位素）和98％L -精氨酸- 13 C 6（0.036克/ L时，剑桥同位素）。 </li><li>诱饵应变定期丰富的培养基中生长正常的同位素氨基酸。 </li></ul></li><li>第一阶段的TAP纯化[此协议是改编自冷泉港实验室课程手册（4）] **您的净化，预寒意迫击炮和杵在-80 ° C冰箱前的夜晚。此外，前寒意一个烧杯-20 ° C ** 之前做出正确的使用这些缓冲区（1升文化）： <ul><li> 30毫升的NP - 40缓冲液25μLPIC（1 / 2000），50μL1M数码地面电视</li><li> 20毫升的NP - 40与1 / 200 PIC，50μL1M数码地面电视的缓冲区</li><li> 10毫升的TEV - C的缓冲与PIC的1 / 2000，10μL的1 M数码地面电视</li></ul></li></ol> SILAC和准备<ol><li>长1L诱饵的应变和控制每一株分别外径600 = 1.0-1.3。 </li><li>倒入500ml中NALGENE离心管文化。 2580X G.平衡负载和颗粒细胞</li><li>倒出媒体和重新悬浮颗粒冷DH 2 O50毫升细胞转移至50ml离心管。沉淀细胞。 **您可以冻结颗粒在-80 ° C ** </li><li> **重要**匹配控制在1:1的诱饵菌种菌种干颗粒重量。 </li><li>重悬NP - 40与1 / / 2,000蛋白酶抑制剂（PIC），缓冲区的每个细胞沉淀于10ml。 </li><li>混合两个细胞培养，直接到其他调迁一管。 研磨 </li><li>倒入液态氮预冷迫击炮，并允许它完全蒸发。加入2毫升细胞砂浆打圈。倒入一些液体氮冻结的细胞。研磨细胞，待细胞成为细粉。重复这一过程，直到所有的细胞是地面。 **不要让解冻的细胞。添加液态氮必要时** </li><li>粉转移到冰冷的烧杯中，在室温下解冻。当边缘开始解冻，1 / 200的PIC添加20毫升NP - 40的缓冲区。 </li><li>转移原油裂解液40毫升NALGENE管。旋转39000 xgfor 30分钟。 </li><li>在等待过程中，采取300μL琼脂糖凝胶2B珠公司（GE）。 NP - 40缓冲液（1 / 2，000 PIC），3次在300μL清洗珠。在300μL缓冲液重悬珠，使他们在1:1水泥浆。 预结算细胞裂解液 </li><li>将上清转移至新管，并添加琼脂糖凝胶2B珠。在30分钟的冷室的旋转平台上孵育。 </li><li>在等待过程中，取300μL的IgG琼脂糖凝胶6珠公司（GE）。 NP - 40缓冲液（1 / 2，000 PIC），3次在300μL清洗珠。重悬在300 _L缓冲区珠，使他们在1:1水泥浆。 </li><li>颗粒的珠子。将上清转移至新管。 **保存Western杂交后的细胞裂解液分装** 绑定到IgG抗体琼脂糖珠 </li><li>取出离心琼脂糖凝胶2B珠。新增的IgG珠（以前1:1浆准备）。为更有效地结合管分为两个独立的内容。在寒冷的房间为2小时的旋转平台上孵育。 </li><li>自旋向下的珠子。 **保存的绑定派等分** </li><li>用10毫升NP - 40缓冲液（1 / 2，000 PIC），珠。 </li><li>洗3毫升TEV - C的缓冲液（1 / 2，000 PIC），珠。 **保存珠等分。这将反映效率约束力** TEV蛋白酶裂解IgG抗体珠洗脱 </li><li>添加5μLAcTEV1毫升TEV - C的缓冲液（1 / 2，000 PIC的）。混合后，分成两个1.5毫升Eppendorf管的内容，更高效混合。在寒冷的房间旋转平台上，孵育过夜。 </li><li>旋珠和洗脱液转移到一个新的1.5 ml管。 TEV - C的缓冲液0.5毫升清洗珠</li><li>结合一管elaute。你应该有共1.5毫升洗脱液。**保存等分的TEV洗脱液** 三氯乙酸（TCA）precipatation（质谱为基础的分析，我们建议使用乙醇/醋酸沉淀） </li><li>调整分装至25％，100％TCA TCA。要做到这一点，分成2管各750μL1.5 ml洗脱液。加入250μL100％的TCA管。您的解决方案应该变成乳白色。 </li><li>置于冰上30分钟周期性振荡。 </li><li>在30分钟的冷房中的表离心机以最大的速度旋转。 </li><li>用500μL冰冷的最高速度（冷室）5分钟的0.05 ñ盐酸和自旋丙酮洗一次。 </li><li>冰冷的丙酮和最大（冷室）5分钟的旋转与500μL洗一次。 </li><li>小心取出丙酮和干燥颗粒。 </li><li>银染法，在50μL1X SDS样品缓冲液重悬沉淀。 乙醇/醋酸precipatation </li><li>加入20微克的糖原</li><li> 5X 100％乙醇稀释样品，并调整与250万的股票，pH值5.0至50毫米NaCH 3 COO </li><li> 2小时的待机解决方案</li><li>颗粒沉淀，离心10 min，室温在12000 X克蛋白质。 </li></ol> NP - 40缓冲液（200毫升） <table border="0"><tbody><tr><td></td><td>股权集中度</td><td>添加</td></tr><tr><td> 10 mM磷酸钠缓冲液（pH值7.2） </td><td> 0.1 M的</td><td> 20毫升</td></tr><tr><td> 150 mM氯化钠</td><td> 2米</td><td> 15毫升</td></tr><tr><td> 1％NP - 40 </td><td> 100％ </td><td> 2毫升</td></tr><tr><td> 50 mM的氟化钠</td><td> 1米</td><td> 10毫升</td></tr><tr><td> 0.1毫米的Na 3 VO 4 </td><td> 10毫米</td><td> 2毫升</td></tr><tr><td style="text-align:right;" colspan="3">容至200 ml </td></tr></tbody></table>在使用前添加： <ol><li> 1 / 1，000 1米的数码地面电视</li><li> 1 / 2，000 PIC（蛋白酶抑制剂的鸡尾酒） </li></ol> TEV - C的缓冲液（50毫升） <table border="0"><tbody><tr><td></td><td>股权集中度</td><td>添加</td></tr><tr><td> 25毫米的Tris（pH值8.0） </td><td> 100毫米</td><td> 12.5毫升</td></tr><tr><td> 150 mM氯化钠</td><td> 2米</td><td> 3.75毫升</td></tr><tr><td> 0.1％NP - 40 </td><td> 100％ </td><td> 50 _L </td></tr><tr><td> 0.5毫米EDTA </td><td> 500毫米</td><td> 50 __l </td></tr><tr><td style="text-align:right;" colspan="3">容至50 ml </td></tr></tbody></table>在使用前添加： <ol><li> 1 / 1，000 1M数码地面电视</li><li> 1 / 2，000的PIC </li></ol>

<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纯化的第一阶段，以尽量减少样品的损失。如果遇到大量的非特异性结合，您可能希望进行的整个协议，可以在冷泉港手册（4）发现。

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 ...

identification-protein-complexes-with-quantitative-proteomics-s

Nanyang Technological University

Research

JoVE Journal

Biology

13.1K 次观看.  University of British Columbia - UBC. 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。

视频: Identification of protein complexes with quantitative proteomics in S. cerevisiae

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

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

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

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

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

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

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

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 ...

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

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

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

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

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

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

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

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

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

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

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

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

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 ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...