| Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria
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Author:
Date:
2017-05-05
[Abstract] The meningococcus (Neisseria meningitidis) remains an important threat to human health worldwide. This Gram-negative bacterium causes elevated disabilities and mortality in infected individuals. Despite several available vaccines, currently there is no universal vaccine against all circulating meningococcal strains (Vogel et al., 2013). Herein, we describe a new protocol that is capable of identifying only cell surface exposed proteins that play a role in immunity, providing this research field with a more straightforward approach to identify novel vaccine targets. Even though N. meningitidis is used as a model in the protocol herein described, this protocol can be used for any Gram-negative bacteria provided modifications and optimizations are carried out to ...
[摘要] 脑膜炎球菌（脑膜炎奈瑟氏球菌）仍然是全球人类健康的重大威胁。这种革兰氏阴性细菌导致感染个体的残疾和死亡率升高。尽管有几种可用的疫苗，目前还没有针对所有循环脑膜炎球菌菌株的通用疫苗（Vogel等人，2013）。在这里，我们描述了一种能够识别仅在细胞表面暴露的蛋白质在免疫中发挥作用的新方案，为该研究领域提供了一种更直接的方法来鉴定新的疫苗靶标。即使使用脑膜炎奈瑟氏球菌作为本文所述方案中的模型，该方案可用于任何革兰氏阴性细菌，提供修饰和优化以使其适应不同的细菌和疾病特征（例如薄膜脆性，生长方法，血清抗体水平，等等）。
背景 尝试开发针对N型的新型疫苗。脑膜炎脑膜炎常常依赖于2D SDS-PAGE（二维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳）和蛋白质印迹，随后MS（质谱）（Wheeler等人，2007））。然而，这种方法采用全细胞裂解物，鉴定出不具有疫苗潜力的大量蛋白质（Mendum等人，2009）。因此，我们旨在开发一种能够鉴别可能在免疫中起重要作用的细胞表面暴露蛋白质的方法。简言之，我们的方案包括生长感兴趣的病原体，用免疫个体的血清免疫沉淀表面抗原，并通过液相色谱 - ...
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| Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
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Author:
Date:
2015-06-05
[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient ...
[摘要] 使用更复杂的显微镜，活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动，趋化性，细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外，细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位，并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物，从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON（Hirst等，2013）。 ...
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| RNA-Affinity Chromatography
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Author:
Date:
2013-07-05
[Abstract] RNA-affinity chromatography assays are used to identify proteins binding specific RNA sequences. These proteins represent potential factors contributing to the function of RNA molecules. In our lab, we have used this protocol to identify proteins binding sequence motifs involved in replication and transcription of positive strand RNA viruses. The assay described in this protocol consists on the immobilization of 5’-biotinylated RNA oligonucleotides (30-40 nt) on a streptavidin-conjugated, paramagnetic solid matrix. Then, cytoplasmic protein extracts pre-cleared on the solid matrix to decrease nonspecific binding, were incubated with the immobilized RNA molecules in the presence of a nonspecific competitor. RNA-protein complexes immobilized on the paramagnetic solid matrix were isolated ...
[摘要] RNA亲和层析分析用于鉴定结合特异性RNA序列的蛋白质。 这些蛋白质代表有助于RNA分子功能的潜在因素。 在我们的实验室，我们使用这个方案来鉴定参与正链RNA病毒复制和转录的蛋白质结合序列基序。 该方案中描述的测定法包括将5'-生物素化的RNA寡核苷酸（30-40nt）固定在链霉抗生物素蛋白缀合的顺磁性固体基质上。 然后，在固体基质上预先清除的细胞质蛋白质提取物以降低非特异性结合，在非特异性竞争剂存在下与固定的RNA分子一起温育。 使用磁铁分离固定在顺磁性固体基质上的RNA-蛋白复合物，并通过聚丙烯酰胺凝胶电泳分离结合蛋白进行蛋白质组学分析。
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