Name: analysis of snare-mediated membrane fusion using an enzymatic cell fusion assay
Uploaded: 2012-10-19T13:00:00
Description: Watch this Scientific Journal Video about Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay at JoVE.com

这项工作是支持的大学，路易斯维尔和CA135123从美国国立卫生研究院（CH）的启动资金。

1。细胞培养和转染<ol><li>补充与4.5克/升葡萄糖和10％牛胎儿血清（FBS）的Dulbecco改良的Eagle氏培养基（DMEM）中培养COS-7细胞。 </li><li>根据制造商的说明（Invitrogen公司），与Lipofectamine质粒转染。 </li></ol> 2。 SNARE细胞表面表达的荧光显微分析<ol><li>转染前一天，3×10 4个 COS-7细胞接种在无菌的12毫米的盖玻片（Fisher Scientific）的24孔培养板中所载。 </li><li>对于v-细胞，在各?...

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原细胞融合实验6荧光显微镜决定SNARE蛋白介导的细胞融合。在这里，我们描述了一个创新的分析，量化SNARE蛋白介导的细胞融合，通过激活β-半乳糖苷酶的表达和光谱测量。使用这个实验中，我们经常分析15 - V-20和T-SNARE组合在一个单一的实验。使用流式细胞仪来测量圈套在细胞表面的表达，我们滴定的鞋面的表达水平，并比较它们的膜融合的能力（图2和图4）。此外，使用的酶的细胞融?...

<table border="1"><tbody><tr><td> 试剂和设备名称 </td><td> 公司 </td><td> 目录号 </td></tr><tr><td> DMEM </td><td> Invitrogen公司</td><td> 12800-017 </td></tr><tr><td> COS-7细胞</td><td> ATCC </td><td> CRL-1651 </td></tr><tr><td>衣霉素</td><td> Sigma-Aldrich公司</td><td> T7765-5MG </td></tr><tr><td> pTet关闭</td><td> Clontech公司</td><td> K1620-A </td></tr><tr><td> PBI-G </td><td> Clontech公司</td><td> 6150-1 </td></tr><tr><td>脂质体转染法</td><td> Invitrogen公司</td><td> 18324-012 </td></tr><tr><td>酶细胞分离解决方案</td><td> Invitrogen公司</td><td> 13151-014 </td></tr><tr><td>兔抗-TET阻遏多克隆抗体</td><td>密理博</td><td> AB3541 </td></tr><tr><td> FITC标记的驴抗小鼠IgG（H + L） </td><td>江淮KSON immunoresearch公司</td><td> 715-095-150 </td></tr><tr><td>激光扫描共聚焦显微镜</td><td>奥林巴斯</td><td> FV1000 </td></tr><tr><td> FACSCalibur流式细胞仪</td><td> BD Biosciences公司</td><td></td></tr><tr><td> β-半乳糖苷酶活性检测系统的报告裂解缓冲液</td><td> Promega公司</td><td> E2000 </td></tr><tr><td>型号100-40 UV-VIS分光光度计</td><td>日立</td><td> C740843 </td></tr></tbody></table>

analysis of snare-mediated membrane fusion using an enzymatic cell fusion assay

T-SNARE蛋白的相互作用的SNARE（oluble N-乙基马来酰亚胺敏感因子一个的重整 ttachment蛋白受体）的目标膜蛋白在囊泡（V-SNARE蛋白）和（）催化细胞内的囊泡融合1-4。重组分析是必不可少的解剖机制和调节SNARE蛋白介导的膜融合5。在细胞融合实验6,7中 ，异位SNARE蛋白表达在细胞表面。这些“翻转”SNARE蛋白驱动细胞与细胞融合，展示，网罗足够细胞膜融合。由于测定是基于微观分析的细胞融合，它是低效率的，使用时，多个的v-叔SNARE相互作用定量分析。 在这里，我们描述了一种新的检测的量化β-半乳糖苷酶的活性表达的SNARE蛋白介导的细胞融合。二在Tet-关基因表达系统9的组件被用作读出系统：四环素控制的反式激活（tTA的）和四环素反应元件（TRE-LacZ的）的控制下，编码的LacZ基因的报道质粒。我们tTA的转染到COS-7细胞表达翻转的v-SNARE蛋白在细胞表面（的v-细胞）和TRE-LacZ基因转染到COS-7细胞表达翻转叔SNARE蛋白在细胞表面（t细胞） 。 SNAP-依赖的融合的v-和T-细胞的查询结果中的结合的tTA的TRE，LacZ和β-半乳糖苷酶的表达的转录激活。 β-半乳糖苷酶的活性，使用的比色的方法通过在420 nm处的吸光度进行定量。 囊泡相关膜蛋白（VAMPS），是V-SNARE蛋白驻留在不同的后高尔基体泡状车厢10-15。鞋面1，3，4，5，7和8，在相同的水平表达，我们比较了它们的膜融合活动中使用的酶的细胞融合实验。基于光谱测量，该法提供了一个量化的方法分析SNARE蛋白介导的膜融合，为高通量研究。

要制定一个定量的细胞融合实验，我们利用强有力的转录激活TTA到TRE的结合。在tTA的的情况下，转录的 LacZ基因在TRE-LacZ的是无声的。 tTA的存在时，它的TRE结合并激活的 LacZ基因的转录， 图1示出了流程图的酶的细胞融合实验。 tTA的转染到v-细胞表达的v-SNARE蛋白在细胞表面，和TRE-LacZ基因转染到t细胞，在细胞表面的表达叔SNARE蛋白。的v-和T-细胞的查询结果中?...

Watch this Scientific Journal Video about Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay at JoVE.com

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

我们已经开发出一种量化SNARE介导膜融合事件由激活的β-半乳糖苷酶的表达的细胞融合实验。

SNARE蛋白介导的膜融合酶的细胞融合含量的分析

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Research

JoVE Journal

Biology

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

科研

生物学

Anterior Cervical Discectomy and Fusion in the Ovine Model

颈椎前路椎间盘切除术和融合在绵羊模型

Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

速度更快，高分辨率，MTPA-GFP基于线粒体融合含量的收购多个单元格的动力学数据，在并行使用共聚焦显微镜

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

在成细胞分化和融合过程中利用活成像跟踪核

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

在尼科蒂亚娜本 thamiana中瞬态表达IgG融合蛋白的生产

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

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睾丸融合的显微外科梗阻

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated ...