| A Blood-retina Barrier Permeability Assay in Young Mice Using Sulfo-NHS-LC-biotin Perfusion
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Author:
Date:
2018-10-20
[Abstract] Brain and retinal vasculatures exhibit restricted vascular permeability known as blood-brain barrier and blood-retina barrier. Vascular permeability can be evaluated by perfusion of the amine reactive ester derivatives of biotin such as sulfo-NHS-LC-biotin. This protocol describes experimental procedures of sulfo-NHS-LC-biotin perfusion to evaluate retinal vascular permeability. Perfused sulfo-NHS-LC-biotin remained within vessels in wild-type postnatal day 15 (P15) retinas, confirming an intact blood-retina barrier. In contrast, sulfo-NHS-LC-biotin was occasionally detected in extravascular spaces in perfused Eogt−/− retinas suggesting a partly impaired vascular integrity in the absence of Eogt (Sawaguchi et al., 2017).
[摘要] 脑和视网膜脉管系统表现出受限的血管通透性，称为血脑屏障和血 - 视网膜屏障。 血管通透性可以通过灌注生物素的胺反应性酯衍生物如磺基-NHS-LC-生物素来评估。 该方案描述了磺基-NHS-LC-生物素灌注的实验程序，以评估视网膜血管通透性。 灌注的磺基-NHS-LC-生物素保留在野生型出生后第15天（P15）视网膜的血管内，证实了完整的血 - 视网膜屏障。 相比之下，在灌注的 Eogt - / - >视网膜中，偶尔会在血管外空间检测到磺基-NHS-LC-生物素，这表明在没有的情况下血管完整性部分受损。 Eogt >（Sawaguchi et al。>，2017）。
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| Dissection and Whole Mount Staining of Retina from Neonatal Mice
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Author:
Date:
2018-10-05
[Abstract] Here we provide a detailed protocol for whole mount staining of mouse retina. This protocol was used to analyze retinal angiogenesis in newborn mice (Sawaguchi et al., 2017) by modifying the original protocols (Powner et al., 2012; Tual-Chalot et al., 2013). This protocol can also be used for whole mount staining of adult retina.
[摘要] 在这里，我们提供了小鼠视网膜整体染色的详细方案。 该方案用于分析新生小鼠的视网膜血管生成（Sawaguchi et al。，2017），修改原始方案（Powner et al。，2012; Tual-Chalot et al。，2013）。 该方案也可用于成人视网膜的整体染色。
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| Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method
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Author:
Date:
2018-09-05
[Abstract] Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine residues facing the hydrophilic environment, even within the transmembrane domain. Cysteine crosslinking experiments provide us with information about the distance between two cysteine residues. The combination of these methods enables us to obtain information about the structural changes of the target protein. Here, we describe the detailed protocol for structural analysis using SCAM.
[摘要] 取代半胱氨酸可及性方法（SCAM）是一种生物化学方法，用于研究目标蛋白中取代的特定半胱氨酸残基的水可及性或空间距离。蛋白质拓扑和结构可以通过用甲硫代磺酸盐试剂标记来注释，所述甲硫基磺酸盐试剂特异性地与面向亲水环境的半胱氨酸残基反应，甚至在跨膜结构域内。半胱氨酸交联实验为我们提供了关于两个半胱氨酸残基之间距离的信息。这些方法的组合使我们能够获得有关靶蛋白结构变化的信息。在这里，我们描述了使用SCAM进行结构分析的详细协议。
【背景】结构分析提供了关于靶蛋白功能的关键信息。 X射线晶体学和核磁共振已被用作生物学领域中的高分辨率蛋白质结构分析方法。然而，这些方法需要以非常高的浓度从膜中提取的纯化蛋白质用于膜蛋白的结构分析。取代半胱氨酸可及性方法（SCAM）是一种生化方法，用于分析目标蛋白中取代的特定半胱氨酸残基的水可及性和空间距离。使用特异性地与面向亲水环境的半胱氨酸残基反应的甲硫代磺酸盐（MTS）试剂，我们可以注释目标蛋白的拓扑结构和结构。由于标记试剂 N - 生物素氨基乙基甲硫基磺酸盐（MTSEA-生物素）对质膜是不可渗透的（Seal et ...
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