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D-(+)-Glucose

D-(+)-葡萄糖

Company: Sigma-Aldrich
Catalog#: G8270
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Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method
Author:
Date:
2020-04-05
[Abstract]  This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (Em = -55 mV) in the SdhA flavoprotein subunit of succinate dehydrogenase from Escherichia coli. In principle, this method can be used routinely to determine the redox potential of flavin cofactors in any simple flavoprotein from equilibrium concentrations with an appropriate reference dye of known Em without the use of sophisticated electrochemical equipment. [摘要]  [摘要] 该协议描述了一种简单的黄嘌呤/黄嘌呤氧化酶酶平衡方法,用于测定黄素的氧化还原电位。作为使用此方法的一个例子,我们确定了大肠杆菌中琥珀酸脱氢酶的SdhA 黄素亚基中共价结合的FAD辅因子的还原电位(E m = -55 mV)。原则上,该方法可常规用于通过平衡浓度和已知E m 的适当参考染料确定任何简单黄素蛋白中黄素辅因子的氧化还原电位。 无需使用复杂的电化学设备。


[背景] 几种生物物理方法可用于测量蛋白质中黄素的还原中点电位(E m )。这些电位测量方法通常依赖于目标蛋白质和电极之间的电化学偶联。例如,在带有其他辅助因子(例如铁硫中心和醌)的复杂黄素蛋白中,常常使用电化学方法和电子顺磁共振(EPR)光谱来确定氧化还原中心的E m (Kowal 等,1995;Saenger等,等人,2005; Hudson 等人,2005; Cheng 等人,2015)。然而,在只包含黄素氧化还原中心简单黄素蛋白,辅助因子,可以直接通过它不需要特殊的设备电化学或昂贵的EPR仪表传统光学分光光度法研究。1990年,文森特·梅西(Vincent Massey)提出了一种简单的方法,该方法可以从氧化和还原伴侣(即黄素蛋白和参考染料)的平衡浓度中确定黄素的还原电位(Massey,1991)。该方法不直接测量黄素的还原电势,而是确定黄素和参考染料的E m ...

Contemporaneous Measurement of Outer and Inner Membrane Permeability in Gram-negative Bacteria
Author:
Date:
2020-03-05
[Abstract]  The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the ... [摘要]  [摘要 ] 细菌中多药耐药性的出现和迅速扩散导致对新型抗菌剂的迫切需求。膜通透性是许多针对革兰氏阴性细菌的抗菌分子的机制。因此,为了确定潜在的抗菌分子的功效,重要的是评估治疗后细菌膜通透性的变化。这项研究描述了使用荧光探针N-苯基-1-萘胺和碘化丙啶测定外膜和内膜通透性的方案。与其他实验(例如电子显微镜和最小杀菌浓度的测定)相比,该方法提供了一种更简单,更快速且经济高效的方法来评估抗菌分子的膜通透性和杀菌功效。这项研究提出了一种相对于传统方案的优化方案,该方案是在与抗菌测定相同的培养条件下,将细菌与抗菌分子一起孵育,然后在不含肉汤和抗菌分子的缓冲液中检测荧光探针的信号。此协议可避免的效果养分缺乏对细菌的生理状态和抗菌molecul的干扰ES 朝向的F luorescent探针。因此,该方法可以有效,准确地评估膜的渗透性,并与从其他抗菌测定法(如最小抑菌浓度和时间杀灭曲线测定法)获得的结果相匹配。

[背景 ] ...

Preparation and Purification of Proteins Secreted from Phytophthora sojae
Author:
Date:
2018-10-20
[Abstract]  Phytophthora sojae, the causal agent of soybean root and stem rot, is responsible for enormous economic losses in soybean production. P. sojae secrets various effectors to reprogram host immunity. The plant apoplastic space is a major battleground in plant-pathogen interactions. Here we describe a protocol for purification and isolation of secreted proteins from P. sojae, including precipitation of secreted proteins from P. sojae culture filtrate, chromatographic purification of the secreted proteins and analysis of the proteins by Mass spectrometry. With this protocol, it will be easier to identify potential apoplastic effectors in Phytophthora and will benefit our understanding of plant-microbe interactions. [摘要]  大豆根和茎腐病的致病因子大豆疫霉(Phytophthora sojae)是造成大豆生产的巨大经济损失的原因。 P上。 sojae >秘密各种效应器重新编程宿主免疫力。植物质外体空间是植物 - 病原体相互作用的主要战场。在这里,我们描述了从 P中纯化和分离分泌蛋白的方案。 sojae >,包括来自 P的分泌蛋白的沉淀。 sojae >培养滤液,分泌蛋白的色谱纯化和质谱分析蛋白质。通过该协议,可以更容易地鉴定 Phytophthora >中潜在的质外体效应物,并有助于我们对植物 - 微生物相互作用的理解。

【背景】从 Phytophthora >物种中分泌的蛋白质的纯化对于理解 Phytophthora >发病机理至关重要。过去,V8果汁和植物(番茄和利马豆)果汁培养基已用于培养 Phytophthora >,培养物过滤后用于分析 Phytophthora >分泌的蛋白质。这些方案的缺点是培养基含有大量的植物蛋白,它们代表了大部分检测到的蛋白质。在该方案中,我们使用了不含任何蛋白质的合成液体培养基。该培养基显着降低了 Phytophthora >培养滤液的背景。此外,利用凝胶过滤脱盐和筛分柱代替离子交换柱,可以有效和大规模纯化 Phytophthora >分泌的蛋白质。

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