Endpoint or Kinetic Measurement of Hydrogen Sulfide Production Capacity in Tissue Extracts
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Author:
Date:
2017-07-05
[Abstract] Hydrogen sulfide (H2S) gas is produced in cells and tissues via various enzymatic processes. H2S is an important signaling molecule in numerous biological processes, and deficiencies in endogenous H2S production are linked to cardiovascular and other health complications. Quantitation of steady-state H2S levels is challenging due to volatility of the gas and the need for specialized equipment. However, the capacity of an organ or tissue extract to produce H2S under optimized reaction conditions can be measured by a number of current assays that vary in sensitivity, specificity and throughput capacity. We developed a rapid, inexpensive, specific and relatively high-throughput method for quantitative detection of H2S ...
[摘要] 通过各种酶法在细胞和组织中产生硫化氢(H 2 S)。 H 2 S是许多生物过程中重要的信号分子,内源性H 2 S生产的缺陷与心血管和其他健康并发症有关。稳态H 2 S水平的定量是由于气体的挥发性和专门设备的需要而具有挑战性的。然而,器官或组织提取物在优化的反应条件下产生H 2 S的能力可以通过许多灵敏度,特异性和通过能力变化的现有测定来测量。我们开发了用于从生物组织定量检测H 2 S生产能力的快速,廉价,特异性和相对高通量的方法。释放到生物样品上方的头部空间中的H 2 S 2与乙酸铅反应形成硫化铅,其使用平板阅读器或作为终点测定法连续测量。 【背景】通过哺乳动物(CGL,CBS,3-MST)中的至少三种不同的酶以一定范围的组织和细胞类型分布内源性地产生硫化氢(H 2 S)。 H 2 S作为与代谢相关的广泛生物学功能的气体发射器和效应分子(Wang,2012)起作用(Módiset al。,2013),应力抵抗(Hine等人,2015)和氧化还原生物学(Dickhout et al。,2012)。减少的H 2 S与心血管问题有关,包括啮齿动物中的高血压(Yang等人,2008)和人心脏肥大(Polhemus等人, ,2014)。增加的H 2 S也可引起病理学,例如啮齿动物胰腺炎(Bhatia et al。,2005)。因此,从生物来源准确和定量地检测H 2 ...
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Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni2+-NTA Plate
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Author:
Date:
2016-03-05
[Abstract] The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant conditions (i.e. in the presence of Zn2+ or with EDTA to chelate Zn2+) and then allowed to compete for binding to S6 which was maintained in limiting concentration. The result was obtained through an ELISA, where S6-His is first bound to a Ni2+-NTA plate, followed by addition of S18-2 in excess to S6, then by addition of increasing concentrations of S18-1. The percentage of S18-2 that remained bound to S6 was ...
[摘要] 设计置换测定以定量来自结核分枝杆菌(Mycobacterium tuberculosis)S18-1和S18-2的两个同源核糖体蛋白质之间的直接竞争,用于与它们的同源结合伴侣,核糖体蛋白S6(Prisic et al。,2015)。 S18蛋白质在两种生理相关条件下(即在Zn 2+ 2+存在下或用EDTA螯合Zn 2+ 2+)进行透析,并且然后允许竞争结合S6,其维持在有限浓度。通过ELISA获得结果,其中S6-His首先结合Ni 2+ 2+ -NTA板,然后加入超过S6的S18-2,然后加入递增浓度的S18-1。在化学发光ELISA中用S18-2蛋白和第二抗体特异性的抗体定量保留结合S6的S18-2的百分比。以这种方式,S18-1蛋白质被S18-1蛋白质的置换报告为通过用S18-2饱和S6实现的全强度信号的百分比。在其基础上,该方法利用天然蛋白质 - 蛋白质相互作用,并且可以应用于其中两种或更多种蛋白质竞争结合上述靶配体的其他系统。
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