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Magnesium Sulfate Heptahydrate (Crystalline/Certified ACS), Fisher Chemical

Company: Fisher Scientific
Catalog#: M63-500
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Extraction and Quantification of Polyphosphate (polyP) from Gram-negative Bacteria
Author:
Date:
2018-09-20
[Abstract]  Polyphosphate (polyP), a universally conserved biomolecule, is composed of up to 1,000 phosphate monomers linked via phosphoanhydride bonds. Reaching levels in bacteria that are in the high nmoles per mg protein range, polyP plays important roles in biofilm formation and colonization, general stress protection and virulence. Various protocols for the detection of polyP in bacteria have been reported. These methods primarily differ in the ways that polyP is extracted and/or detected. Here, we report an improved method, in which we combine polyP extraction via binding to glassmilk with a very sensitive PolyP kinase/luciferase-based detection system. By using this procedure, we significantly enhanced the sensitivity of polyP detection, making it potentially applicable for mammalian tissues. [摘要]  多磷酸盐(polyP)是一种普遍保守的生物分子,由多达1,000个通过磷酸酐键连接的磷酸盐单体组成。 达到每毫克蛋白质高纳摩尔细菌的水平,polyP在生物膜形成和定植,一般应力保护和毒力中起重要作用。 已经报道了用于检测细菌中polyP的各种方案。 这些方法主要在于提取和/或检测polyP的方式不同。 在这里,我们报告了一种改进的方法,其中我们结合polyP提取通过结合到玻璃奶与非常敏感的PolyP激酶/荧光素酶检测系统。 通过使用该程序,我们显着增强了polyP检测的灵敏度,使其可能适用于哺乳动物组织。

【背景】聚磷酸盐(polyP)是一种由多达1,000种无机磷酸盐单体的直链组成的生物聚合物,存在于生命的所有三个领域的细胞中。然而,细菌是唯一已经充分研究了polyP代谢酶的生物。将ATP转化为polyP的细菌polyP激酶(PPK)催化正向和反向反应。虽然polyP的合成显然是细胞中有利的反应,但通过在体外提供足够量的ADP ,该酶可用于从polyP产生ATP,使得基于荧光素酶的ATP检测成为可能(Ault -Riché et al。,1998)。缺乏PPK的细菌在生物膜形成,运动性,持久性和各种应激反应方面存在缺陷,并显示出对次卤酸(即,漂白)应激或磷酸盐饥饿的显着增加的敏感性(图1)(Rao et al。,2009; Gray et ...

Quantification of Bacterial Twitching Motility in Dense Colonies Using Transmitted Light Microscopy and Computational Image Analysis
Author:
Date:
2018-04-20
[Abstract]  A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified by the degree to which the bacteria modulated the intensity of light in the field-of-view over time. This allowed the mapping of areas of relatively high and low motility within a single field-of-view, and comparison of the total distribution of motility between samples. [摘要]  开发了一种方法,可以对密集样本中的相对细菌抽动动力进行定量和绘图,在这些样本中追踪单个细菌是不可行的。 在这种方法中,使用微分干涉对比显微镜(DIC)获得细菌膜的电影,然后通过细菌随时间调节视场中的光强度的程度间接量化细菌运动。 这允许在单个视场内绘制相对较高和较低运动性的区域,并比较样本之间运动的总分布。

【背景】Pilus介导的颤动运动表示与鞭毛无关的与表面相关的细菌运动形式。抽动动力被很多细菌病原体利用,包括淋病奈瑟氏球菌和铜绿假单胞菌与潮湿的表面相互作用并移位上皮屏障。在 P。颤动动力受大量基因调控,这些基因允许IV型菌毛的延伸和回缩,以有效地将细菌细胞拖过任何给定的表面以响应环境提示(Mattick,2002; Whitchurch et al。,2004; Burrows,2005)。在我们对 P的研究中。绿脓杆菌发病机制,抽动运动性有助于细菌在内化和多层角膜上皮细菌穿过后从上皮细胞排出(Alarcon等人,2009)。在角膜感染的小鼠模型中,抽动运动对于P是重要的。绿脓杆菌毒力(Zolfaghar et al。,2003)。最近,我们发现在粘膜液体如人眼泪和唾液中发现的糖蛋白DMBT1能够抑制P细胞。绿脓杆菌抽动动力(Li等人,2017)。在那项研究中,我们利用了一种新方法来快速和可靠地量化P.绿脓杆菌抽动动力。该协议在此处介绍。
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Measurement of Lysosomal Size and Lysosomal Marker Intensities in Adult Caenorhabditis elegans
Author:
Date:
2018-02-05
[Abstract]  Assays have been developed to study trafficking in various tissues of Caenorhabditis elegans. Adult C. elegans intestinal cells are large and have extensive endocytic networks, thus making them a good system for deciphering the endocytic pathway using live imaging techniques. However, the presence of auto-fluorescent gut granules in adult intestine can interfere with the signals of endocytic compartment reporters, like GFP. Here we demonstrate a protocol adapted from the original method developed by the Grant laboratory to identify signals from reporters in adult intestinal cells. The goal of this protocol is to identify endocytic compartments tagged with fluorescent markers without any confounding effects of background autofluorescent gut granules in adult intestinal ... [摘要]  已经开发了用于研究线虫的各种组织中的贩运的测定法。 成人 C。 线虫肠细胞很大,具有广泛的内吞网络,因此使它们成为使用实时成像技术破译内吞途径的良好系统。 然而,成年肠道中存在自身荧光的肠道颗粒会干扰吞噬细胞记者的信号,如GFP。 在这里,我们展示了一个协议,从格兰特实验室开发的原始方法改编,以确定记者在成人肠细胞的信号。 该方案的目标是识别标记有荧光标记物的内吞室,而在秀丽隐杆线虫成虫肠细胞中没有背景自发荧光消化道颗粒的混杂效应。

【背景】秀丽隐杆线虫(Caenorhabditis elegans)是一种多细胞生物,已被用于研究内吞运输。最初开发了用于研究内吞的试验。线虫卵母细胞,胚胎和体细胞(清道夫细胞)。简而言之,卵母细胞和胚胎中的测定通过在逗号至1.5倍发育阶段测量肠隔室中含有蛋黄 - 蛋白 - 绿色荧光蛋白报道分子(VIT-2 :: GFP)的区室的强度和大小来进行Grant和Hirsh,1999; Schaheen等人,2006a)。在成年人中,在转基因成虫的体细胞中测量了含有GFP的隔室的强度和大小(从体壁肌细胞分泌进入囊泡并由体细胞胞吞)。表达肌肉myo-3 :: ...

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