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吐温 20

Company: Sigma-Aldrich
Catalog#: P9416
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Microfluidics-Based Analysis of Contact-dependent Bacterial Interactions
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Date:
2018-08-20
[Abstract]  Bacteria in nature live in complex communities with multiple cell types and spatially-dependent interactions. Studying cells in well-mixed environments such as shaking culture tubes or flasks cannot capture these spatial dynamics, but cells growing in full-fledged biofilms are difficult to observe in real time. We present here a protocol for observing time-resolved, multi-species interactions at single-cell resolution. The protocol involves growing bacterial cells in a near monolayer in a microfluidic device. As a demonstration, we describe in particular observing the dynamic interactions between E. coli and Acinetobacter baylyi. In this case, the protocol is capable of observing both contact-dependent lysis of E. coli by A. baylyi via the Type VI ... [摘要]  自然界中的细菌生活在具有多种细胞类型和空间依赖性相互作用的复杂社区中。 在充分混合的环境中研究细胞,例如摇动培养管或烧瓶,不能捕获这些空间动力学,但是在完全成熟的生物膜中生长的细胞难以实时观察。 我们在这里提出了一种用于观察单细胞分辨率下的时间分辨,多物种相互作用的方案。 该方案涉及在微流体装置中以近单层生长细菌细胞。 作为演示,我们特别描述了观察 E之间的动态相互作用。 大肠杆菌和 Acinetobacter baylyi 。 在这种情况下,该方案能够观察 E的接触依赖性裂解。 大肠杆菌由 A。 baylyi 通过VI型分泌系统(T6SS)和随后的来自 E的基因的功能性水平基因转移(HGT)。 大肠杆菌到 A.baylyi。

【背景】 不同种类细菌之间的空间依赖性相互作用可能在自然界普遍存在,但它们很难观察到。一个例子是通过天然感受的捕食性DNA受体细胞的接触依赖性,原位裂解作为DNA供体的捕食细胞来增强水平基因转移(HGT)。这种情况最近才出现在革兰氏阴性菌中,但已经在多个物种中观察到,并且它被认为是一种相对普遍的现象(Borgeaud et al。,2015; Cooper 等人,,2017; Veening和Blokesch,2017; Ringel et ...

Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry
Author:
Date:
2018-07-05
[Abstract]  Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure ... [摘要]  来自产后啮齿动物的小脑颗粒神经元(CGN)已被广泛用作研究神经元发育,生理学和病理学的模型。 CGN体外培养维持成熟小脑颗粒细胞在体内显示的相同特征,包括发育致密的神经炎网络,神经元活动,神经递质释放和神经元的表达 蛋白质标记。 此外,CGN代表了梭菌神经毒素(CNT)研究的便利模型,最着名的是破伤风和肉毒杆菌神经毒素,因为它们大量表达CNT受体和神经元内基质, ie ,可溶性N-乙基马来酰亚胺 - 敏感因子激活蛋白受体(SNARE蛋白)。 在这里,我们描述了从出生后大鼠/小鼠获得高纯度CGN培养物的方案和用CNT中毒的简便方法。 我们还说明了评估CNT活性及其抑制的方便方法。

【背景】梭菌神经毒素(CNT)的大家族由破伤风神经毒素(TeNT)和肉毒杆菌神经毒素(BoNT)的多种变体形成,它们分别是破伤风和肉毒中毒的神经麻痹毒素(Schiavo et al。,2000; Johnson和Montecucco,2008; Rossetto et al。,2014)。 TeNT,七种BoNT血清型(BoNT / A至/ G)及其许多亚型是金属蛋白酶,通过切割SNARE蛋白(可溶性N-乙基马来酰亚胺敏感因子激活蛋白受体),三种必需蛋白质来阻断神经递质的释放而引起神经麻痹。控制突触小泡与突触前质膜的融合(Rossetto et al。,2014; ...

In vitro Analysis of Ubiquitin-like Protein Modification in Archaea
Author:
Date:
2018-05-20
[Abstract]  The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of Ubl protein modification and following analysis of this modification in Haloferax volcanii, a halophilic archaeon serving as the model organism. [摘要]  泛素样(Ubl)蛋白广泛分布于古细菌中并参与许多细胞途径。 为了更好地理解古细菌Ub1蛋白质修饰的过程,重建体外古细菌Ubl蛋白质缀合物的完善方法是很重要的。 该协议描述了Ubl蛋白质修饰的体外重建以及在作为模型生物的嗜盐古细菌Haloferax volcanii 中对这种修饰进行分析。

【背景】泛素(Ub)与靶蛋白共价连接的过程被称为泛素化,其控制真核细胞中大量的细胞过程(Glickman和Ciechanover,2002; Komander和Rape,2012)。遍在蛋白化由一系列酶(包括Ub激活酶(E1),Ub结合酶(E2s)和Ub连接酶(E3s))催化。泛素化的体外重建是确定酶之间或E3与蛋白质底物之间特异性的有用测定法(Zhao等人,2012)。在古细菌中,Ubl蛋白SAMP采用Ub折叠,并且与E1样酶UbaA催化的蛋白靶标异肽连接[Maupin-Furlow,(2014)综述]。尽管E1同系物在古细菌中广泛存在,但基于一级序列比较,在大多数古细菌中未预测经典E2或E3酶。我们最近对Haloferax volcanii的研究表明甲硫氨酸亚砜还原酶A(MsrA)是Ubl蛋白质修饰(sampylation)与UbaA一起在体内温和的氧化条件下和< (体外)(fu="">

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