Name: detection of toxin translocation into the host cytosol by surface plasmon resonance
Uploaded: 2012-01-03T13:00:00
Description: Watch this Scientific Journal Video about Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance at JoVE.com

这项工作是由美国国立卫生研究院授予R01 AI073783光Teter。我们感谢援助，发展批判性阅读的手稿中的亚细胞分馏协议和海伦布瑞斯博士谢恩梅西。

1。对毛地黄皂苷的制备<ol><li>离心管加入500μL100％的乙醇，并将其放置在在80℃10分钟的热块。 </li><li>溶解在加热乙醇250μL2.5毫克毛地黄皂苷产生的毛地黄皂苷1％原液。 </li><li>要产生0.04％毛地黄皂苷的工作液，添加40μL毛地黄皂苷原液HCN的缓冲液960μL（50 mM的HEPES pH值7.5，150 mM氯化钠，氯化钙2 2毫米，10毫米的N - ethylmaleimide，和一种蛋白酶抑制剂鸡尾酒）。 </li></ol> 2。?...

与现有方法的比较我们基于SPR -易位检测的一种快速，敏感，定量的方法来检测毒素进入宿主细胞质交付。该技术并不需要放射性标记或其他修改的毒素，它可以应用到任何毒素，抗毒素A链抗体可。监测到细胞质的毒素通过现有的方法还依赖于一个分区成单独的细胞质和膜组分11，23，27，28的细胞提取物的亚细胞分馏协议。这些方法使用免疫印迹或radiolabels检测胞浆内?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>毛地黄皂苷</td><td>西格玛</td><td> D141 </td></tr><tr><td>乙醇</td><td>阿库罗斯福冈</td><td> 61509-0010 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11995065 </td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11550 </td></tr><tr><td>神经节苷脂</td><td>西格玛</td><td> G7641 </td></tr><tr><td>注册税务师</td><td>西格玛</td><td> C2398 </td></tr><tr><td> PTS1 </td><td>名单</td><td> 182 </td></tr><tr><td> NHS（N -羟基） </td><td>刺穿</td><td> 24500 </td></tr><tr><td> EDC（1 - 乙基-3 - （3 - dimethylaminopropyl）亚胺） </td><td> Thermo Scientific的</td><td> 22981 </td></tr><tr><td>乙醇胺</td><td>泗GMA </td><td> E0135 </td></tr><tr><td> PBST </td><td>紫花苜蓿</td><td> 09-8903-100 </td></tr><tr><td>反CTA抗体</td><td>圣克鲁斯生物技术</td><td> SC - 80747 </td></tr><tr><td>反城巴抗体</td><td> Calbiochem </td><td> 227040 </td></tr><tr><td>抗PTS1抗体</td><td>圣克鲁斯生物技术</td><td> SC - 57639 </td></tr><tr><td>折射</td><td>赖克特</td><td> SR7000，SR7000DC </td></tr><tr><td> SPR传感器幻灯片</td><td>赖克特</td><td> 13206060 </td></tr><tr><td>注射泵</td><td>科尔帕尔默</td><td> 780200C </td></tr></tbody></table>

detection of toxin translocation into the host cytosol by surface plasmon resonance

AB毒素组成的酶的A亚单位和一个细胞结合B 亚基 1 。这些毒素分泌到细胞外的环境，但他们的行为于真核细胞质内的目标。有些AB毒素旅游载体囊泡从细胞表面的内质网（ER），才进入细胞质2-4。在急诊室，不赞成从其余的毒素和移动通过一种蛋白质，在进行渠道的催化A链，以达到其胞质目标 5 。易位，胞质链是难以察觉，因为毒素贩运到急诊室是一个非常低效的过程：路由到最内在毒素降解的溶酶体，所以只有一小部分的表面结合的毒素达到高尔基体和ER 6 -12。 为了监视从ER在培养细胞的细胞质中的毒素易位，我们结合highl亚细胞分馏协议y的表面等离子体共振（SPR）13-15敏感的检测方法。毒素处理细胞的细胞膜选择性通透与毛地黄皂苷，使收集的一个细胞质部分，这是对涂有抗毒素A链抗体的SPR传感器随后灌注。抗体包传感器，可以捕捉和检测pg / mL的胞浆内毒素的数量。有了这个协议，它是可以遵循的毒素进入到细胞质的动力学特征易位事件的抑制作用。胞浆内毒素的浓度也可以从已知数量的A链已灌注在传感器的标准生成标准曲线计算。我们的方法代表了一种快速，敏感，定量检测系统不需要放射性标记或其他修改目标毒素。

Watch this Scientific Journal Video about Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance at JoVE.com

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

在这份报告中，我们描述了表面等离子体共振是如何用来检测输入到宿主细胞质中的毒素。胞浆内毒素量，这个高度敏感的方法可以提供定量数据，并可以应用到一系列的毒素。

表面等离子体共振检测到宿主细胞质的毒素易位

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

Research

JoVE Journal

Immunology and Infection

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

通过横向流技术检测在血液系统恶性肿瘤患者侵袭性肺曲菌病

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

免疫荧光共聚焦显微镜对单纯疱疹病毒1蛋白核细胞质转移的时间分析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...