| Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification
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Author:
Date:
2018-11-20
[Abstract] Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer’s. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the functions of Tau and the generation of pharmacological agents targeting Tau has been the difficulty of obtaining soluble Tau protein in purified form. Here, we describe a protocol that uses dual affinity tag purification to selectively purify soluble recombinant Tau protein from bacteria that is functionally active for downstream applications including immunization, microtubule binding assays, and protein-protein interaction studies.
[摘要] 微管相关蛋白Tau（由 MAPT 基因编码）的功能障碍已经涉及20多种神经退行性疾病，包括阿尔茨海默病。 因此，Tau的生理和疾病相关功能引起了研究界的极大兴趣。 妨碍对Tau功能的研究和产生靶向Tau的药理学试剂的一个障碍是难以获得纯化形式的可溶性Tau蛋白。 在这里，我们描述了一种方案，该方案使用双亲和标签纯化从细菌中选择性纯化可溶性重组Tau蛋白，所述细菌对于下游应用具有功能活性，包括免疫，微管结合测定和蛋白质 - 蛋白质相互作用研究。
【背景】Tau传统上被定义为微管结合蛋白;然而，在人类疾病中，Tau可以与轴突微管分离并错误定位到其他神经元区室，包括体细胞，树突和突触，其中与非微管蛋白和结构的相互作用驱动神经元功能障碍（Iqbal et al。 ，2016; Wang和Mandelkow，2016; Zhou et al。，2017; McInnes et al。，2018）。尽管神经原纤维缠结形式的Tau聚集体通常存在于死后患病的脑组织中，但研究表明，可溶性Tau，而不是聚集的Tau，是神经元功能障碍的主要原因（Crimins et al。，2012 ; Polydoro et al。，2014; Koss et al。，2016）。因此，研究Tau在疾病中的可溶性功能，例如鉴定蛋白质 - ...
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| Detection of Cell Death in Planarians
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Author:
Date:
2018-10-05
[Abstract] Planarians are freshwater flatworms, well known for their ability to regenerate a complete organism from any piece of their body. Furthermore, planarians are constantly growing and degrowing throughout their lives, maintaining a functional and proportioned body. These properties rely on the presence of a population of adult stem cells and on the tight control of their cell renewal, which is based on the balance between the proliferation of new cells and their differentiation, and the death of unnecessary cells. Due to the importance of these two processes in planarian biology, over the years, researchers have optimized molecular techniques to detect both cell proliferation and cell death in planarians. Here, we present the two main protocols currently used for cell death detection and ...
[摘要] 涡虫是淡水扁虫，因其能够从身体的任何部分再生完整的有机体而闻名。 此外，涡虫在其一生中不断生长和去除，保持功能和比例的身体。 这些特性依赖于成体干细胞群的存在以及对细胞更新的严格控制，其基于新细胞增殖与其分化之间的平衡以及不必要细胞的死亡。 由于这两个过程在涡虫生物学中的重要性，多年来，研究人员已经优化了分子技术，以检测涡虫中的细胞增殖和细胞死亡。 在这里，我们提出了目前用于细胞死亡检测和量化的两种主要方案：Caspase-3活性定量和TUNEL分析。
【背景】成体生物体中的细胞更新是基于三个过程的复杂机制：（a）通过细胞死亡消除选定的细胞; （b）通过细胞分裂取代已消除的细胞，通常涉及成体干细胞及其后代; （c）新生细胞的分化及其与先前存在的组织的整合（Pellettieri和Sanchez ...
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| Isolating Liver Mitochondria by Differential Centrifugation
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Author:
Date:
2016-05-20
[Abstract] In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice liver tissue. Mitochondria obtained with this method can be used to assess different mitochondrial parameters, including oxygen consumption, membrane potential and calcium retention capacity.
[摘要] 除了旨在研究线粒体功能的方法之外，充分了解线粒体功能需要在特定生理或病理条件下从细胞或组织中纯化。 该方案说明从小鼠肝组织获得高产量的功能性线粒体的顺序程序。 用该方法获得的线粒体可用于评估不同线粒体参数，包括氧消耗，膜电位和钙保留能力。
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