Name: chromatographic purification of highly active yeast ribosomes
Uploaded: 2011-10-24T12:00:00
Description: Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

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?...

<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. A. 70, 3866-3869 (1973).</li><li>Nathans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Puromuycin+inhibition+of+protein+synthesis:+incorporation+of+puromycin+into+peptide+chains.">Puromuycin inhibition of protein synthesis: incorporation of puromycin into peptide chains.</a> Proc. Natl. Acad. Sci. U. S. A. 51, 585-592 (1964).</li><li>Rabinovitz, M., Fisher, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dissociative+effect+of+puromycin+on+the+pathway+of+protein+synthesis+by+Ehrlich+ascites+tumor+cells.">A dissociative effect of puromycin on the pathway of protein synthesis by Ehrlich ascites tumor cells.</a> J. Biol. Chem. 237, 477-481 (1962).</li><li>Allen, D. W., Zamecnik, 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=The+effect+of+puromycin+on+rabbit+reticulocyte+ribosomes.">The effect of puromycin on rabbit reticulocyte ribosomes.</a> Biochim. Biophys. Acta. 55, 865-874 (1962).</li><li>Yarmolinsky, M. B., Haba, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+by+puromycin+of+amino+acid+incorporation+into+protein.">Inhibition by puromycin of amino acid incorporation into protein.</a> Proc. Natl. Acad. Sci. U. S. 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>

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.  ...

Research

JoVE Journal

Biology

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 ...

科研

生物学

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 ...

Preparation of Highly Coupled Rat Heart Mitochondria

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

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

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

In vitro Reconstitution of the Active T. castaneum Telomerase

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

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, ...

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 ...

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

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

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 ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

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 ...