Name: monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein
Uploaded: 2012-11-10T14:00:00
Description: Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

这项研究由国家老化（NFN S93）由奥地利科学基金会（FWF），欧盟委员会综合项目MiMAGE和PROTEOMAGE，荷兰基因组计划研究网络/荷兰科学研究组织（NGI / NWO; 05040202，050 - 060-810恩查），欧盟资助网络的卓越寿命（FP6 036894），和基因组学创新研究计划（SenterNovem IGE01014和IGE5007）。

1。质粒的构建<ol><li>订购定制寡核苷酸编码为DGN（ACKNWFSSLSHFVIHL 11）和为dgnFS（HARTGSLACPTSSSICE）和结扎到质粒pEGFP-C1载体，以获得与DGN / dgnFS的（ 图1）的GFP的融合。 </li><li>通过PCR扩增的GFP-dgn和GFP-dgnFS的的编码序列，。根据协议pENTR定向TOPO克隆试剂盒，并继续与pLenti6/V5定向TOPO克隆试剂盒（ 图6）。 </li></ol> 2。病毒生产<ol><li>转染前一天（第1天?...

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用绿色荧光蛋白（GFP）作为底物泛素-蛋白酶体活性记者，发表于2000年12首次出版。从那时起，GFP已成为一种普遍的工具，可视化细胞的活动，特别是泛素 - 蛋白酶体的过程。若要监视在体内的泛素-蛋白酶体活性的转基因小鼠模型与基于GFP的记者已被引入13。额外另一种转基因小鼠模型的体内研究建立一个类似的不稳定degron的GFP记者作为本出版物中的14。 <p...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> PEGFP-C1载体</td><td> BD生物科学Clontech公司</td><td> 6084-1 </td></tr><tr><td> pENTR定向TOPO克隆试剂盒</td><td> Invitrogen公司</td><td> K2400-20 </td></tr><tr><td> pLenti6/V5定向TOPO克隆试剂盒</td><td> Invitrogen公司</td><td> V496-10 </td></tr><tr><td>脂质体转染试剂</td><td> Invitrogen公司</td><td> 11668019 </td></tr><tr><td> DMEM </td><td>西格玛</td><td> D5546 </td></tr><tr><td> PVDF过滤器（Rotilabo-Spritzenfilter） </td><td>罗斯</td><td> P667.1 </td></tr><tr><td>聚乙二醇</td><td>西格玛</td><td> P2139 </td></tr><tr><td>氯化钠</td><td>默克</td><td> 1.06404.1000 </td></tr><tr><td> Dulbecco磷酸盐缓冲贝东北1X（PBS） </td><td> Invitrogen公司</td><td> 14190 </td></tr><tr><td>溴化己二甲铵溴化</td><td>西格玛</td><td> 10,768-9 </td></tr><tr><td>杀稻瘟</td><td> Invitrogen公司</td><td> R21001 </td></tr><tr><td>结晶紫</td><td>西格玛</td><td> C3886 </td></tr><tr><td> FACS管</td><td> BD Biosciences公司</td><td></td></tr><tr><td>青霉素链霉素（笔链球菌） </td><td> Invitrogen公司</td><td> 15140130 </td></tr><tr><td> L-谷氨酰胺200mM的</td><td> Invitrogen公司</td><td> 25030024 </td></tr><tr><td>胎牛血清（FBS） </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM非必需氨基酸（NEAA）100X </td><td> Invitrogen公司</td><td> 11140035 </td></tr><tr><td> MEM丙酮酸钠100毫米</td><td> Invitrogen公司</td><td> 11360039 </td></tr><tr><td> D-（+）葡萄糖（45％） </td><td>西格玛</td><td> G8769 </td></tr><tr><td>标记基因</td><td> Invitrogen公司</td><td> 11811023 </td></tr><tr><td>氯化钙</td><td>默克</td><td> C5080 </td></tr><tr><td> HEPES </td><td>西格玛</td><td> H3375 </td></tr><tr><td>胰蛋白酶-EDTA（0.05％） </td><td> Invitrogen公司</td><td> 25300054 </td></tr></tbody></table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

蛋白酶是异常的，错误折叠的，损坏或氧化蛋白1，2中的蛋白水解降解的主要涉及的胞内细胞器。蛋白酶体活性的维护被牵连了许多关键的细胞过程，如细 ​​胞的应激反应，细胞周期调控和细胞分化或免疫系统的反应5。已与泛素-蛋白酶体系统的功能障碍，肿瘤，神经退行性疾病4，6的发展。此外，在蛋白酶体活性的减少被发现作为一个功能的细胞衰老和有机体的老化7，8，9，10。在这里，我们提出了一种方法来衡量使用GFP-的DGN融合蛋白在活细胞的泛素 - 蛋白酶体的活性。至能够监视泛素 - 蛋白酶体活性的活的原代细胞，互补的DNA构建体编码的绿色荧光蛋白（GFP）-DGN融合蛋白（GFP-DGN，不稳定）和携带的移码突变的变体（GFP-dgnFS的，稳定11）被插入在慢病毒表达载体。我们更喜欢这种技术比传统的转染技术，因为它保证了一个非常高的转染效率的细胞类型或捐赠者的年龄无关。可以使用显示在GFP-dgnFS（稳定）蛋白和蛋白酶体抑制剂的存在或不存在中的不稳定的蛋白（GFP-DGN）的荧光之间的差异，来估计在每个特定的细胞株的泛素 - 蛋白酶体活性。这些差异可以通过荧光显微镜监测，也可以通过流式细胞仪检测。

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

监控活细胞的泛素 - 蛋白酶体活性的方法。一个degron不稳定的绿色荧光蛋白（GFP-DGN）和一个稳定的的GFP-dgnFS融合蛋白的生成和使用慢病毒表达载体导入细胞。这种技术允许产生稳定的GFP-dgn/GFP-dgnFS表达细胞株中，泛素 - 蛋白酶体活性可以很容易地使用的反射荧光或流式细胞术评估。

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Research

JoVE Journal

Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

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 ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...