Name: purification and visualization of influenza a viral ribonucleoprotein complexes
Uploaded: 2009-02-09T00:00:00
Description: Watch this Scientific Journal Video about Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes at JoVE.com

<p class="jove_content">这项工作是由加拿大创新基金会（CFI），加拿大卫生研究所（CIHR）学院，以及自然科学与工程研究理事会（NSERC），加拿大的赠款支持。 </p>

<p class="jove_title">第1部分：中断一个流感病毒粒子</p><ol><li>添加到一个贝克曼聚碳酸酯离心管，设计成TLA - 120.2转子适合在贝克曼的Optima使用（11毫米× 34毫米）的750名产妇和新生儿破伤风缓冲液（MES的20毫​​米，150毫米氯化钠，30毫米的Tris，pH值7.5）马克斯 - E超速离心机。 </li><li>添加到该管的A型流感病毒（H3N2型X - 31 A/AICHI/68应变; 2毫克/毫升）500μL。移液器向上和向下几次与产妇和新生儿破伤风的缓冲区混合病毒...

<ol>
	<li>Kemler, I., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+microinjected+influenza+virus+ribonucleoproteins.">Nuclear import of microinjected influenza virus ribonucleoproteins.</a> <em style='font-style: italic'>Virology</em>. <b>202</b>, 1028-1033 (1994).</li><li>Wu, W. W. H., Weaver, L. L., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+the+nuclear+localization+sequences+on+influenza+a+ribonucleoprotein+complexes.">Ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes.</a> <em style='font-style: italic'>J Mol Biol</em>. <b>374</b>, 910-916 (2007).</li><li>Gasteiger, E., Walker, 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> <em style='font-style: italic'>The Proteomics Protocols Handbook</em>. , 571-607 (2005).</li><li>Wu, W. W. H., Sun, Y. H. B., Pant&#233;, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+influenza+A+viral+ribonucleoprotein+complexes+is+mediated+by+two+nuclear+localization+sequences+on+viral+nucleoprotein.">Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein.</a> <em style='font-style: italic'>Virol J</em>. <b>4</b>, 49-49 (2007).</li><li>Babcock, H. P., Chen, C., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-particle+tracking+to+study+nuclear+trafficking+of+viral+genes.">Using single-particle tracking to study nuclear trafficking of viral genes.</a> <em style='font-style: italic'>Biophys J</em>. <b>87</b>, 2749-2758 (2004).</li></ol>

<p class="jove_content"> vRNPs净化Kemler <em>等人</em> （1994年）所描述的程序的基础上，我们和其他人也用此协议隔离vRNPs，研究它们<sup>的</sup>核进口。2,4,5 </p><p class="jove_content">我们建议使用无RNase的技巧和操纵vRNPs管时，因为病毒基因组RNA的组成，因此很容易降解的RNA的存在。此外，所有的缓冲区，应即核糖核酸免费的水。这里所描述的协议，是一种酸性提取（如中断缓冲区和甘油梯度的pH值是5.5）。同样，一个基本的?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>H3N2 X-31 A/AICHI/68 influenza A</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>490715</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M-8250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G33-1</td>
<td>&nbsp;</td>
<tr>
<td>Octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O-8001</td>
<td>&nbsp;</td>
<tr>
<td>Lysolecithin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4129</td>
<td>&nbsp;</td>
<tr>
<td>Dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-9779</td>
<td>&nbsp;</td>
<tr>
<td>Coomassie brilliant blue G-250</td>
<td>&nbsp;</td>
<td>Kodak</td>
<td>1367796</td>
<td>&nbsp;</td>
<tr>
<td>diethyl pyrocarbonate (DEPC)-treated water</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>750024</td>
<td>&nbsp;</td>
<tr>
<td>Uranyl Acetate</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>19481</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Molybdate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A-674</td>
<td>&nbsp;</td>
<tr>
<td>Optima MAX-E Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>434491</td>
<td>&nbsp;</td>
<tr>
<td>MLS-50 Rotor Package, Swinging Bucket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>367280</td>
<td>&nbsp;</td>
<tr>
<td>TLA-120.2 Rotor Assembly, Fixed-Angle, Titanium</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362046</td>
<td>&nbsp;</td>
<tr>
<td>Eppendorf Thermomixer</td>
<td>&nbsp;</td>
<td>Brinkman</td>
<td>022670000</td>
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purification and visualization of influenza a viral ribonucleoprotein complexes

<p class="jove_content">A型流感病毒的基因组由8负意义上说，单链RNA分子单独包装，与A型流感到的病毒ribonulceoprotein颗粒（vRNPs）核蛋白（NP）的多个副本。流感vRNPs封闭的病毒包膜内。然而，在进入细胞，这些vRNP复合物释放到细胞质，在那里他们获得主机核运输机械。为了研究核进口的流感vRNPs和流感基因组复制的，它与孤立的vRNPs工作，使病毒的其他组件不干扰这些进程是有益的。在这里，我们描述一个过程来净化这些vRNPs从A型流感病毒。程序启动以释放笼罩病毒粒子vRNP复合物，用清洁剂A型流感病毒粒子的干扰。 vRNPs是从A型流感病毒粒子在33-70％的速度沉淀间断甘油梯度的其他部分隔开。从甘油梯度获得的分数，然后通过SDS - PAGE电泳分析，考马斯亮蓝染色后。包含NP峰值分数，然后汇集在一起​​，并通过离心浓缩。浓度后，vRNPs的完整性是通过可视化的传输电子显微镜负染色后的vRNPs验证。甘油梯度纯化的修改，从Kemler<em&gt;等。</em&gt;（1994）<sup&gt; 1</sup&gt;和负染色进行吴<em&gt;等。</em&gt;（2007年）。<sup&gt; 2</sup</p>

Watch this Scientific Journal Video about Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes at JoVE.com

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

<p class="jove_content">A型流感病毒的基因组由8个独立的复合物，RNA和蛋白质，称为病毒核蛋白复合物（vRNPs）。本文介绍的甘油梯度纯化和透射电子显微镜A型流感vRNPs可视化。</p>

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

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

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

制备，纯化和磁共振成像造影剂的使用稀土配合物的表征

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

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.  ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in <em>Escherichia coli</em> 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 ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

对链球菌-Tactin树脂交联的双（磺基）辛二酸酯（BS3）双链球菌标记蛋白及其复合物的一步纯化

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

产生和人类INO80染色质重塑复合物及其亚纯化

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...