我们衷心感谢Dinman实验室的所有成员，包括阿什顿Belew，莫文蔚杰克，Sharmishtha Musalga，谢尔盖Sulima，Shivani雷迪，他们的帮助和在这个项目上的投入和Michael Rhodin。这项工作得到了补助金，上午由美国心脏协会（AHA 0630163N）和开发部的国立卫生研究院（5R01 GM058859 - 12）。日航是支持美国再投资和恢复法案2009年补充父补助金（3R01GM058859 11S1）。

1。半胱氨酸负责筹备Sulfolink树脂<ol><li>准备Sulfolink树脂（皮尔斯）在室温下。 </li><li>广场共10毫升50％的Sulfolink耦合两个10毫升的塑料瓶制造商提供的凝胶浆，分发到每个小瓶5毫升。 </li><li>短暂离心瓶850 XG，小心取出上清液。 </li><li>耦合缓冲冲洗凝胶3次（50毫米三，pH值8.5，5毫米EDTA）resuspending在5毫升的珠子，离心850简要XG和消除吸取上清液。 </li><li>每管添加50毫米的L -半胱氨酸耦合缓冲溶液5毫升，混合浆料1小时25 ° C。 </li><li>删除残留洗涤步骤1.4以上所述的L -半胱氨酸。 </li><li> 10毫升结合缓冲液重悬凝胶[20毫米的HEPES，pH值7.6，5毫米镁（OAC）2，60毫米NH 4 CL，2毫米的数码地面电视]，和平衡的结合缓冲液10毫升冲洗树脂中所述的步骤1.4的3倍。倒出树脂到10 ml离心柱，帽和保存在4 ° C。为RNA结合树脂能力是〜20每毫升260个单位。 </li></ol> 2。使用半胱氨酸核糖体的色谱净化收取Sulfolink树脂注：如果与蛋白酶的活性，证明是一个问题的应变工作，可在随后的所有缓冲区使用蛋白酶抑制鸡尾酒。 <ol><li>保持冰的所有组件，并准备使用无RNase水和无菌过滤所有的解决方案。 </li><li>一夜之间日志中期阶段（外径595 ​​= 0.6 - 1.2），在适当的媒体成长的酵母细胞。离心沉淀细胞，并洗为5分钟1克细胞结合缓冲液，并在3700 XG离心机在4 ° C。 </li><li>悬浮细胞在结合缓冲液1毫升约2毫升的总量，并扰乱使用0.5毫米的玻璃珠等体积冷却到4 ° C U唱一个Biospec迷你珠打浆机（巴特尔斯维尔，确定）。样品可以根据需要分割成多个管。 </li><li>在离心30分钟，在一个贝克曼 - 库尔特舰最大发送超速离心机（富勒顿，加利福尼亚州）30,000 XG取出完整的细胞，细胞器和细胞碎片。 </li><li>使用前一分钟，颗粒树脂在1000 XG删除的存储解决方案。两个毫升树脂是用​​于一克的每一个细胞。 </li><li>删除从30,000 XG自旋（S30）的细胞裂解液上清，直接将收取的Sulfolink浆照顾，以尽量减少污染脂质的一小部分的顶部或在管底部的细胞碎片，并将其放置。脂质层，可避免通过去除或推从吸管尖后上清液越过。 </li><li>在冰上孵育S30/Sulfolink混合搅拌15分钟。 </li><li>放入15 m​​l锥形管列在1000 XG离心机v一个分钟。 </li><li>入列，用手拌匀，并孵化了15分钟在冰上将流出液组分。 </li><li>放入15 m​​l锥形管的列，并在1000 XG离心一分钟。舍弃流出液。 </li><li>第列和结合缓冲液中添加5毫升。手工混合列至重悬，取下镜头盖，并在一分钟1000 XG列离心机离心机。重复此洗涤协议两次。 </li><li>洗脱缓冲液1.5毫升（20毫米HEPES - KOH，pH值7.6，10毫米MG（OAC）2，氯化钾500毫米，2mm的数码地面电视，0.5毫克/毫升肝素），每一列。手工混合的泥浆，并在冰上孵育2分钟</li><li>进入新的15 ml锥形管和离心机的地点列在1000 1分钟XG。收集洗脱液。重复洗脱步骤，再次使最终合并后的样品量〜3毫升。的树脂可与洗脱缓冲液洗涤，无肝素和平衡约束力buffeR，并存储在10毫升卷的结合缓冲液在4 ° C，再以后使用。 </li></ol> 3。嘌呤霉素治疗注：另一种方法，以消除污染tRNA的物种是从富含葡萄糖葡萄糖耗尽的媒体，以促进核糖体径流开关。然而，这并不改变的酵母细胞，这可能会影响核糖体的功能和浓度的代谢状态。 为了地带核糖体从内源性肽酰- tRNA，与治疗嘌呤霉素进行。 <ol><li>中性pH值7.5 100毫米嘌呤的解决方案，在室温下加入1M氢氧化钾滴加。 </li><li>新增瓦解嘌呤解决〜1mm的终浓度洗脱液。 </li><li> 100毫米的GTP到终浓度为1毫米。 </li><li>孵育30分钟，30 ° C。 </li></ol> 4。通过甘油垫子沉淀净化核糖体<ol><li>放置一个毫升缓冲垫成4毫升量聚碳酸酯超速离心机管（20毫米的HEPES，pH值7.6，10毫米镁（OAC）2，500毫米氯化钾，2毫米数码地面电视，25％甘油）。 </li><li>轻轻层嘌呤霉素治疗垫顶部，洗脱组分离心样品在10万XG 4℃过夜° C。 </li><li>删除离心机转子管，吸出上清液。 </li><li>两次纯化的核糖体含有1毫升缓冲液洗颗粒。 </li><li>在100温柔中断使用玻璃棒的气垫缓冲液重悬核糖体。 </li><li>中断后，用封口膜管，并在一个适中的速度在寒冷的房间涡旋摇一小时。 </li><li>离心管，离心5分钟以最大的速度在4 ° C，传输的内容和新鲜管去除上清。 </li><li>重复步骤4.1至4.7 2毫升的缓冲垫，除了存储缓冲区100μL（50毫米的HEPES - KOH pH值7.6，50 mM的NH 4 CL，5毫米镁（OAC）2，1毫米DTT，25％的甘油）是用来代替气垫缓冲步骤4.4和4.5 。 </li><li>量化纯化的核糖体分光光度计（1一260 = 20 pmoles酵母核糖体），并储存于-80 ° C。从酵母1克的典型结果是300-400核糖体pmoles。 </li></ol> 5。代表性的成果： 从每个协议的三个主要步骤提取的RNA物种的一个例子是如图2所示。虽然rRNAs的主要品种目前在总细胞裂解物（T）的这些还含有大量的其他RNA物种。从Sulfolink列（SL）纯化的核糖体，也包含了这些物种的高柱床的亲和力以及由于大量的tRNA。从核糖体治疗，这在从肽酰- tRNA的多肽水解嘌呤结果的一小部分，并促进这些物种的解离。嘌呤霉素治疗的样本（下午）缺乏合作purifyin克tRNA的物种，从而代表完全纯净的核糖体。据此前报道8，这些核糖体是高度完整和生化活跃，使他们详细的功能和结构分析的理想衬底。 <img src="/files/ftp_upload/3214/3214fig1.jpg" alt="图1"/> 图1。层析纯化方法的流程图。使用挂钩sulfolink树脂的半胱氨酸核糖体描绘。 <img src="/files/ftp_upload/3214/3214fig2.jpg" alt="图2"/> 图2。代表分析核糖体的筹备工作。总RNA的物种分别提取总细胞裂解物（T）Sulfolink纯化的核糖体（SL），并随后与嘌呤霉素治疗Sulfolink纯化的核糖体，并通过甘油垫（下午）沉淀。巷M代表RNA的大小标记。乐队代表25S和18S rRNAs，以及tRNA基因和其他RNA物种表示。所有车道装有2微克的RNA。

<ol>
	<li>Palade, G. E., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+microsomes;+an+integrated+morphological+and+biochemical+study.">Liver microsomes; an integrated morphological and biochemical study.</a> J. Biophys. Biochem. Cytol. 2, 171-200 (1956).</li><li>Fabry, M., Kalvoda, L., Rychlik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interactions+of+Escherichia+coli+ribosomes.">Hydrophobic interactions of Escherichia coli ribosomes.</a> Biochim. Biophys. Acta. 652, 139-150 (1981).</li><li>Jelenc, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+highly+active+ribosomes+from+Escherichia+coli.">Rapid purification of highly active ribosomes from Escherichia coli.</a> Anal. Biochem. 105, 369-374 (1980).</li><li>Le goffic, F., Moreau, N., Chevelot, L., Langrene, S., Siegrist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+bacterial+ribosomes+using+chloramphenicol+and+erythromycin+columns.">Purification of bacterial ribosomes using chloramphenicol and erythromycin columns.</a> Biochimie. 62, 69-77 (1980).</li><li>Meskauskas, A., Petrov, A. N., Dinman, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+functionally+important+amino+acids+of+ribosomal+protein+L3+by+saturation+mutagenesis.">Identification of functionally important amino acids of ribosomal protein L3 by saturation mutagenesis.</a> Mol. Cell Biol. 25, 10863-10874 (2005).</li><li>Algire, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+reconstituted+yeast+translation+initiation+system.">Development and characterization of a reconstituted yeast translation initiation system.</a> RNA. 8, 382-397 (2002).</li><li>Maguire, B. A., Wondrack, L. M., Contillo, L. G., Xu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chromatography+system+to+isolate+active+ribosomes+from+pathogenic+bacteria.">A novel chromatography system to isolate active ribosomes from pathogenic bacteria.</a> RNA. 14, 188-195 (2008).</li><li>Leshin, J. A., Rakauskaite, R., Dinman, J. D., Meskauskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purity,+activity+and+structural+integrity+of+yeast+ribosomes+purified+using+a+general+chromatographic+method.">Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method.</a> RNA. Biol. 7, 354-360 (2010).</li><li>Triana, F., Nierhaus, K. H., Chakraburtty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+RNA+binding+to+80S+ribosomes+from+yeast:+evidence+for+three+sites.">Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites.</a> Biochem. Mol. Biol. Int. 33, 909-915 (1994).</li><li>Azzam, M. E., Algranati, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+puromycin+action:+fate+of+ribosomes+after+release+of+nascent+protein+chains+from+polysomes.">Mechanism of puromycin action: fate of ribosomes after release of nascent protein chains from polysomes.</a> Proc. Natl. Acad. Sci. U. S. 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A. 45, 1721-1729 (1959).</li><li>Becker-Ursic, D., Davies, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+phosphorylation+of+ribosomal+proteins+by+protein+kinases+from+Saccharomyces+cerevisiae.">In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.</a> Biochemistry. 15, 2289-2296 (1976).</li></ol>

没有利益冲突的声明。

核糖体净化协议主要涉及裂解细胞，收获一个细胞质分数从低转速旋转，然后造粒核糖体由高速离心1。虽然提出了一些新颖的方法已被用于净化细菌核糖体，同样没有被真正的真核生物2-4。虽然这些年来，例如盐清洗和甘油垫子（如看到5）增加额外的步骤，酵母核糖体的生化和结构的研究已阻碍了他们的倾向，成为在净化过程中的内源性降解酶降解，最有可能由于长期?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Sulfolink耦合树脂</td><td>刺穿</td><td> 20402 </td></tr><tr><td>迷你珠打浆机16 </td><td> Biospec </td><td> 607 </td></tr><tr><td>舰最大发送超速离心机</td><td> Beckman Coulter公司</td><td></td></tr><tr><td>联想RT </td><td> Sorvall </td><td></td></tr><tr><td> 0.5毫米的玻璃珠</td><td> Biospec </td><td> 11079105 </td></tr><tr><td>三</td><td> Sigma - Aldrich公司</td><td> 252859 </td></tr><tr><td> EDTA </td><td> Sigma - Aldrich公司</td><td> E9884 </td></tr><tr><td>数码地面电视</td><td> Sigma - Aldrich公司</td><td> 43815 </td></tr><tr><td> HEPES </td><td> Sigma - Aldrich公司</td><td> 54459 </td></tr><tr><td> NH 4 CL </td><td> Sigma - Aldrich公司</td><td> A9434 </td></tr><tr><td>氯化钾</td><td> Sigma - Aldrich公司</td><td> 60128 </td></tr><tr><td>镁（OAC）2 </td><td> Sigma - Aldrich公司</td><td> M5661 </td></tr><tr><td>氢氧化钾</td><td> Sigma - Aldrich公司</td><td> 221473 </td></tr><tr><td>甘油</td><td> Sigma - Aldrich公司</td><td> G5516 </td></tr><tr><td>嘌呤霉素</td><td> Sigma - Aldrich公司</td><td> P7255 </td></tr><tr><td> GTP </td><td> Fermentas公司</td><td> R0461 </td></tr></tbody></table>

chromatographic purification of highly active yeast ribosomes

真核生物核糖体相比，他们真细菌和archael更加不稳定，从而构成了重大挑战，研究人员。特别麻烦的是，细胞溶解释放大量的蛋白酶和核酸，这可以降低核糖体。因此，重要的是尽可能快地从这些酶中分离出来的核糖体。不幸的是，传统的差分离心方法叶时间不可接受的长期接触这些酶的核糖体，影响其结构的完整性和功能。为了解决这个问题，我们利用一个色谱法使用收费Sulfolink树脂半胱氨酸。这个简单，快速的应用显着降低水解蛋白和nucleolytic活动，共同净化生产完好，高度生化活性酵母核糖体的高产量。我们建议，这种方法也应适用于哺乳动物的核糖体。简约的方法，增强的纯度和色谱纯化的核糖体的活动的一个显著的技术进步，为真核细胞的核糖体的研究。

Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

Chromatographic Purification of Highly Active Yeast Ribosomes

准备用传统的方法，纯化共同净化核酸酶和蛋白酶的真核细胞的核糖体的污染造成了消极影响下游的生化和结构分析。一个快速和简单的色谱的净化方法是用来解决这个问题作为一个模型系统中使用的酵母核糖体。

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.  ...

chromatographic-purification-of-highly-active-yeast-ribosomes

Research

JoVE Journal

Biology

16.7K 次观看.  University of Maryland. 准备用传统的方法，纯化共同净化核酸酶和蛋白酶的真核细胞的核糖体的污染造成了消极影响下游的生化和结构分析。一个快速和简单的色谱的净化方法是用来解决这个问题作为一个模型系统中使用的酵母核糖体。

视频: Chromatographic Purification of Highly Active Yeast Ribosomes

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 ...

科研

生物学

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 ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

高度耦合的大鼠心肌线粒体的制备

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

分离翻译含肽- tRNA的核糖体的功能和结构分析

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母殖民地嵌入方法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

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 ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...