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Ethanol 70% (v/v) TechniSolv®, pure

Company: VWR
Catalog#: 83801.360
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Detection of Protein Interactions in the Cytoplasm and Periplasm of Escherichia coli by Förster Resonance Energy Transfer
Author:
Date:
2018-01-20
[Abstract]  This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in Escherichia coli by Förster Resonance Energy Transfer (FRET). The described assay allows for the previously impossible in vivo screening of periplasmic protein-protein interactions. In FRET, excitation of a donor fluorescent molecule results in the transfer of energy to an acceptor fluorescent molecule, which will then emit light if the distance between them is within the 1-10 nm range. Fluorescent proteins can be genetically encoded as fusions to proteins of interest and expressed in the cell and therefore FRET protein-protein interaction experiments can be performed in vivo. Donor and acceptor fluorescent protein fusions are constructed for bacterial proteins ... [摘要]  该协议的开发是通过Förster共振能量转移(FRET)定性和定量检测大肠杆菌中的蛋白质 - 蛋白质相互作用。所描述的测定允许以前不可能的周质蛋白质 - 蛋白质相互作用的体内筛选。在FRET中,供体荧光分子的激发导致能量转移到受体荧光分子,如果它们之间的距离在1-10nm范围内,则受体荧光分子将发光。荧光蛋白质可以被遗传编码为与感兴趣的蛋白质的融合物并且在细胞中表达,因此FRET蛋白质 - 蛋白质相互作用实验可以在体内进行。供体和受体荧光蛋白融合体被构建用于被怀疑相互作用的细菌蛋白质。这些融合蛋白在细菌细胞中共表达,随后激发供体和受体通道测量荧光发射光谱。供体的发射光谱与受体的激发光谱之间的部分重叠是FRET的先决条件。即使在没有FRET的情况下,供体激发也可以使受体以已知百分比交叉激发。通过测量背景,仅供体和仅受体样品的参考光谱,可以计算预期的发射光谱。在预期光谱之上的受体的致敏发射可以归因于FRET,并且可以通过光谱解混来量化。

【背景】确定如何和哪些蛋白质相互作用维持生命是分子生物学研究的核心。存在许多体外方法,但可能导致误报,因为相互作用是从其生物学背景中取出的。 ...

Protease Activity Assay in Fly Intestines
Author:
Date:
2017-09-20
[Abstract]  The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the assessment of intestinal functions encompasses, among others, the monitoring of its integrity, its cellular renewing, its immune defenses, the production of enteroendocrine hormones and its digestive capacities. Here, we describe in detail how to assess the activity of the proteases secreted in the intestinal lumen of adult Drosophila melanogaster flies. This method can also be used for larval intestines. The present protocol is adapted and ... [摘要]  肠是消化食物所需的中枢器官,吸收营养物质,并与食物一起摄入的侵略者作斗争。 粘膜损伤后的肠道生理损伤影响其消化能力,从而影响个体的生长,健康甚至生存。 因此,肠功能的评估包括监测其完整性,细胞更新,免疫防御,肠内分泌激素的产生及其消化能力。 在这里,我们详细描述如何评估分泌在成年果蝇的肠腔中的蛋白酶的活性。 这种方法也可以用于幼虫肠。 本协议由“蛋白酶荧光检测试剂盒”(产品代码PF0100)中提出的Sigma-Aldrich公司的方案进行了改进和改进。
【背景】肠道受到诸如斋戒,禁食,化学物质,病原体,损伤等诸多压力的影响。肠道能够通过保持其体内平衡的生理平衡来克服这种压力。为了感知进入的压力并产生适应性的维持肠功能的答案,肠已经开发出强健和保守的机制,如局部先天免疫防御和组织再生(Royet和Charroux,2013; Bonfini等,2016)。然而,在某些情况下,肠道稳态的维持可能受到影响。例如,在老化期间,随着许多未成熟或分化不良的细胞的存在,组织稳态维持总体下降(Jasper,2015; ...

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