| Cloud-point PEG Glass Surfaces for Imaging of Immobilized Single Molecules by Total-internal-reflection Microscopy
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Author:
Date:
2016-04-05
[Abstract] This effective, robust protocol generates glass coverslips coated with biotin-functionalized polyethylene glycol (PEG), making the glass surface resistant to non-specific absorption of biomolecules, and permitting immobilization of biomolecules for subsequent single-molecule tracking of biochemical reactions. The protocol can be completed in one day, and the coverslips can be stored for at least 1 month. We have confirmed that the PEG surfaces prepared according to the protocol are resistant to non-specific adsorption by a wide range of biomolecules (bacterial, mitochondrial, and human transcription factors, DNA, and RNA) and biological buffers.
[摘要] 这种有效的，稳健的方案产生涂覆有生物素官能化聚乙二醇（PEG）的玻璃盖玻片，使得玻璃表面对生物分子的非特异性吸收具有抗性，并允许固定生物分子以用于随后的单分子跟踪生化反应。 协议可以在一天内完成，盖玻片可以存储至少1个月。 我们已经证实，根据方案制备的PEG表面可以通过广泛的生物分子（细菌，线粒体和人转录因子，DNA和RNA）和生物缓冲液的非特异性吸附抗性。
【背景】研究生物分子动力学的单分子成像方法通过允许实时跟踪多步反应而不需要同步试剂来补充传统的“大量”生物化学方法（Weiss，1999）。在大多数单分子成像方法中，首先用单个荧光团标记感兴趣的生物分子，然后将标记的生物分子固定在光学透明的表面（通常为玻璃或二氧化硅）上，并且被检测为衍射受限图像（“斑点”）使用配有高灵敏度相机的光学显微镜（Selvin和Ha，2008）。表面固定有两个目的。首先，它允许在超过几百毫秒的时间尺度上跟踪分子态（否则，生物分子将扩散到焦平面外）。第二，表面允许在全内反射几何中激发荧光（Axelrod，1981），其显着地增加了与表面接近（<100nm）位置的分子的检测的信噪比（selvin和哈，2008）。尽管有这些明显的优势，表面也是单分子分析中最常见的人为因素（visnapuu et=""> ...
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| Thioglycollate-elicited Peritoneal Macrophages Preparation and Arginase Activity Measurement in IL-4 Stimulated Macrophages
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Author:
Date:
2015-09-05
[Abstract] Macrophages are an essential cell population of innate immunity that plays important roles in inflammatory processes. Two main different phenotypes have been described with opposing activities: The classically activated macrophages (M1) and the alternatively activated macrophages (M2). Alternative activation of mouse macrophages can be induced by type 2 cytokines such as IL-4 and it is characterized by the regulation of the L-arginine metabolism. M2 macrophages convert arginine to ornithine and urea through the action of Arginase-1. Here we described a method for the isolation of peritoneal macrophages from thioglycollate-elicited mice and alternative activation by stimulation with IL-4. Intraperitoneal injection of thioglycollate elicits large numbers of macrophages into peritoneal ...
[摘要] 巨噬细胞是在炎症过程中起重要作用的先天免疫的必需细胞群体。 已经描述了具有相反活性的两种主要不同表型：经典激活的巨噬细胞（M1）和交替激活的巨噬细胞（M2）。 小鼠巨噬细胞的备选激活可以由2型细胞因子例如IL-4诱导，并且其特征在于L-精氨酸代谢的调节。 M2巨噬细胞通过精氨酸酶-1的作用将精氨酸转化为鸟氨酸和尿素。 在这里我们描述了从巯基乙酸诱发小鼠分离腹膜巨噬细胞和IL-4刺激的替代激活的方法。 腹膜内注射巯基乙酸盐引发大量巨噬细胞进入腹膜腔。
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| Measurement of Net High-affinity Urea Uptake in Maize Plants
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Author:
Date:
2015-06-05
[Abstract] Despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. So far, the physiological and molecular aspects of urea acquisition have been investigated only in a few plant species highlighting the importance of a urea transporter in roots, DUR3 (Kojima et al., 2007; Wang et al., 2012; Zanin et al., 2014a). Regarding maize plants, a crop that needs a large amount of urea fertilizer, the capability to take up urea via an inducible and high-affinity transport system has been recently characterized (Zanin et al., 2014a; Zanin et al., 2014b). Here, we described a small-scale protocol suitable for the measurement of urea net high-affinity uptake in roots of ...
[摘要] 尽管其作为氮肥的广泛使用，但是作为直接可及的作物植物氮源的脲的作用仍然知之甚少。 迄今为止，仅在少数植物物种中研究了尿素获取的生理学和分子学方面，突出了根中的尿素转运蛋白DUR3的重要性（Kojima等人，2007; et al。，2012; Zanin et al。，2014a）。 关于玉米植物，需要大量尿素肥料的作物，通过诱导型和高亲和力转运系统摄取尿素的能力最近已被表征（Zanin等人，2014a; Zanin et al。，2014b）。 在这里，我们描述了一个小规模的协议，适合测量尿素净高亲和力摄取完好的玉米植物的根。
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