| Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography
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Author:
Date:
2017-04-20
[Abstract] Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of Rs by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ binding, but likely arises from changes in the hydration shell structure.
This ...
[摘要] 尺寸排阻色谱法（SEC）或凝胶过滤是一种流体动力学技术，它将溶液中的分子作为其尺寸和形状的函数。在蛋白质的情况下，可以通过实验得出的流体动力学值是斯托克斯半径（R s），它是具有相同流体力学性质的球体的半径（即， >摩擦系数）作为生物分子。通过SEC测定R s已经被广泛用于监测由钙（Ca 2+）与许多Ca 2+连接引起的构象变化传感器蛋白。对于这类蛋白质，SEC分离不仅基于Ca 2 + 结合后的蛋白质尺寸变化，而且可能来自水合壳结构的变化。
该方案旨在使用快速蛋白质液相色谱（FPLC）系统对预填充柱进行凝胶过滤实验，以确定蛋白质的R 1，其中一些适用于Ca 2 + 传感器蛋白。
凝胶过滤基于其相对的能力分离不同大小和形状的分子，以穿透具有明确孔径的多孔珠床，其识别分馏范围。大于完全排除进入孔隙的分馏范围的分子快速流过色谱柱，首先以空间体积（V 0 O）（其为支持颗粒外的间隙体积）洗脱。能够扩散到珠的孔中的小于分级范围的分子具有可用于流动相的总体积，因此它们更缓慢地移动通过床并最后洗脱。具有中等维度的分子将以包含在流动相可利用的空隙体积和总体积之间的洗脱体积（V e e e e）被洗脱（分子越小，其进入孔隙越大矩阵，因此其V e e越大）。
蛋白质的分子量可以通过比较其洗脱体积参数K ...
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| Quantitative Analysis of Exosome Secretion Rates of Single Cells
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Author:
Date:
2017-02-20
[Abstract] To study the inhomogeneity within a cell population including exosomes properties such as exosome secretion rate of cells and surface markers carried by exosomes, we need to quantify and characterize those exosomes secreted by each individual cell. Here we develop a method to collect and analyze exosomes secreted by an array of single cells using antibody-modified glass slides that are position-registered to each single cell. After each collection, antibody-conjugated quantum dots are used to label exosomes to allow counting and analysis of exosome surface proteins. Detailed studies of exosome properties related to cell behaviors such as responses to drugs and stress at single cell resolution can be found in the publication (Chiu et al., 2016).
[摘要] 为了研究细胞群体内的不均匀性，包括外来体特征，如外来体外分泌细胞分泌速度和外来体携带的表面标志物，我们需要量化和表征每个细胞分泌的那些外来体。在这里，我们开发收集和分析由单个细胞阵列分泌的外来体的方法，使用位置登记到每个单细胞的抗体修饰的玻片。每次收集后，使用抗体缀合的量子点来标记外来体以允许外来体表面蛋白的计数和分析。在出版物（Chiu等人，2016）中可以找到与细胞行为相关的外来物质性质的详细研究，例如对药物的反应和单细胞分辨率的压力。
背景 ...
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| Whole-mount Enteroid Proliferation Staining
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Author:
Date:
2016-06-20
[Abstract] Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology in vitro as they recapitulate the various epithelial cells within the intestinal crypt (Mahe et al., 2013; Sato et al., 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations in gene expression, treatment with protective and toxic agents, and genetic mutations alter properties essential for crypt growth and survival as well as the stem cell properties of the individual cells within the crypt. This protocol describes a method of visualization of proliferating cells within the crypt in three dimensions (Barrett et al., 2015). Whole-mount proliferation staining of enteroids ...
[摘要] 小肠类器官，也称为肠袢，已经成为越来越多地用于肠道生物学的体外模型，因为它们重现了在肠隐窝内的各种上皮细胞（Mahe等人 ，2013; Sato et al。，2009）。在这些培养物中评估生长动力学是理解基因表达的改变，用保护性和毒性试剂处理以及遗传突变改变隐窝生长和存活所必需的性质以及细胞内单个细胞的干细胞性质的重要步骤地穴。该方案描述了在隐窝内三维的增殖细胞的可视化方法（Barrett等人，2015）。使用EdU掺入的肠衣的整体增殖染色使得研究者能够观察到肠内的所有增殖细胞，而不是如在包埋和切片中所见到的在薄切片中获得生长信息，确保来自干细胞区室的增殖的真实表现到隐窝的终末分化细胞。
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