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Chloramphenicol

氯霉素

Company: Sigma-Aldrich
Catalog#: C0378
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Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale
Author:
Date:
2020-04-05
[Abstract]  Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. ... [摘要]  [摘要 ] 人体肠道菌群中的玫瑰菌属和真细菌属在维持人类健康中起着重要作用,部分原因是产生丁酸盐,这是我们结肠上皮细胞的主要能源。但是,由于缺乏基因操作技术,我们对这些细菌的生物化学和生理学的认识受到限制。先前引入玫瑰花属物种的共轭转座子不容易被修饰,极大地限制了它们作为基因修饰平台的适用性。MOD ular质粒穿梭载体先前已经开发了用于梭菌物种,其与共享一个分类次序ř oseburia 和真杆菌,提高这些矢量可以在这些生物体中使用的可能性。在这里,我们描述了一种优化缀合协议使得能够自主复制的质粒的从转印大肠杆菌供体菌株为罗斯氏inulinivorans 和真杆菌rectale 。质粒的模块性质及其通过自主复制在受体细菌中得以维持的能力使其成为研究异源基因表达的理想之选,并成为其他遗传工具(包括反义RNA沉默或II 型移动子中断子基因破坏策略)的平台。

[背景 ] 玫瑰菌和真细菌属人类肠道菌群中含量最高的细菌(Zhernakova 等,2016),它们通过利用饮食和宿主衍生的多糖影响人类健康(Scott 等,2006和2011; Cockburn 等) 。,2015 ; 谢里登等人,2016 )并产生促进健康的代谢物丁酸作为发酵终产物(邓肯等人,2002和2006) 。另外,这些物种能够通过鞭毛调节宿主免疫(Neville ...

Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification
Author:
Date:
2018-11-20
[Abstract]  Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer’s. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the functions of Tau and the generation of pharmacological agents targeting Tau has been the difficulty of obtaining soluble Tau protein in purified form. Here, we describe a protocol that uses dual affinity tag purification to selectively purify soluble recombinant Tau protein from bacteria that is functionally active for downstream applications including immunization, microtubule binding assays, and protein-protein interaction studies. [摘要]  微管相关蛋白Tau(由 MAPT 基因编码)的功能障碍已经涉及20多种神经退行性疾病,包括阿尔茨海默病。 因此,Tau的生理和疾病相关功能引起了研究界的极大兴趣。 妨碍对Tau功能的研究和产生靶向Tau的药理学试剂的一个障碍是难以获得纯化形式的可溶性Tau蛋白。 在这里,我们描述了一种方案,该方案使用双亲和标签纯化从细菌中选择性纯化可溶性重组Tau蛋白,所述细菌对于下游应用具有功能活性,包括免疫,微管结合测定和蛋白质 - 蛋白质相互作用研究。
【背景】Tau传统上被定义为微管结合蛋白;然而,在人类疾病中,Tau可以与轴突微管分离并错误定位到其他神经元区室,包括体细胞,树突和突触,其中与非微管蛋白和结构的相互作用驱动神经元功能障碍(Iqbal et al。 ,2016; Wang和Mandelkow,2016; Zhou et al。,2017; McInnes et al。,2018)。尽管神经原纤维缠结形式的Tau聚集体通常存在于死后患病的脑组织中,但研究表明,可溶性Tau,而不是聚集的Tau,是神经元功能障碍的主要原因(Crimins et al。,2012 ; Polydoro et al。,2014; Koss et al。,2016)。因此,研究Tau在疾病中的可溶性功能,例如鉴定蛋白质 - ...

α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Author:
Date:
2018-07-20
[Abstract]  Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors (e.g., aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive fluorescence reporter of amyloid fibril formation and are conducted in a plate-reader-based format, permitting the simultaneous screening of multiple samples and conditions. However, aggregation assays are generally prone to poor reproducibility due to the stochastic nature of fibril nucleation and the multiplicity of modulating factors. Here we present a simple and reproducible protocol to study the aggregation of α-synuclein in a plate-reader based assay. [摘要]  研究淀粉样蛋白如α-突触核蛋白体外聚集是一种方便和流行的工具,可以获得聚集的动力学见解以及研究因子(例如,聚集抑制剂) 影响它。 这些聚集测定通常利用荧光染料硫磺素T作为淀粉样蛋白原纤维形成的敏感荧光报告物,并且以基于读板器的形式进行,允许同时筛选多个样品和条件。 然而,由于原纤维成核的随机性质和调节因子的多样性,聚集测定通常倾向于较差的再现性。 在这里,我们提出了一个简单和可重复的协议,以研究基于读板器的测定中α-突触核蛋白的聚集。

【背景】内源性蛋白质与淀粉样原纤维的聚集是一种致病过程,与几种疾病相关,例如,神经退行性疾病如阿尔茨海默病(AD)或帕金森病(PD)以及全身性疾病如AL淀粉样变性( Knowles et al。,2014)。通过基于硫磺素T荧光的聚集测定,可以在基于板读者的装置中在体外中概括该过程,从而允许根据各种影响因素研究淀粉样蛋白的聚集动力学。

硫磺素T(ThT)是一种荧光染料,最初用于组织学样本中的淀粉样蛋白原纤维染色,于1959年由Vassar和Culling(Vassar和Culling,1959),其在体外检测和定量淀粉样纤维的应用

目前,硫代黄素T的聚集测定主要在荧光板读数器中进行,其中例如,96条件可以同时测试。由于原纤维成核的随机性质和影响蛋白质聚集的多种因素,这些测定法具有较差的重现性。因此,已经采用了增加ThT测定的再现性的策略,例如在测量期间使用井板的轨道摇动以及向孔中添加玻璃珠以改善混合(Giehm和Otzen,2010)。 ...

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