| 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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| Isolation of Neutralizing Antibody
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Author:
Date:
2013-12-20
[Abstract] Use of monoclonal antibodies (MAbs) is an established laboratory strategy for characterization of specific pathogens and their antigenicity. Especially, Human MAbs (HuMAbs) with neutralizing activity against specific virus could have potential therapeutic application, and provide significant information on human epitopes that could be important for developing the next generation of universal vaccines against the virus. In addition to the classical method for murine MAb preparation, several methods for the preparation of HuMAbs have been developed. Here, we describe the development of neutralizing HuMAbs against specific virus. HuMAbs are established by fusion of the peripheral blood mononuclear cells of vaccinated volunteers or patients with the fusion partner cell line, named SPYMEG. ...
[摘要] 使用单克隆抗体（MAbs）是用于表征特定病原体及其抗原性的已建立的实验室策略。 特别地，具有针对特定病毒的中和活性的人MAb（HuMAb）可具有潜在的治疗应用，并提供对于可能对于开发下一代针对该病毒的通用疫苗重要的人表位的重要信息。 除了用于鼠MAb制备的经典方法外，已经开发了几种制备HuMAbs的方法。 在这里，我们描述中和HuMAbs针对特定病毒的发展。 HuMAbs通过将接种的志愿者或患者的外周血单核细胞与融合伴侣细胞系（称为SPYMEG）融合而建立。 然后确认每个制备的HuMAb是否可以通过体外中和测定中和特异性病毒。
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