| Immunocytochemical Detection of Recombinant Biomphalysin on Schistosoma mansoni Sporocysts
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Author:
Date:
2013-11-20
[Abstract] Schistosomiasis, or bilharzia, is a tropical disease caused by worms of the genus Schistosoma which infect about 200 million people. The life cycle of the parasite requires Biomphalaria, a specific genus of freshwater snails, as intermediate. Using an interactome approach employing B. glabrata plasma and S. mansoni primary sporocyst extracts, we identified a new cytolytic protein called Biomphalysin that displays similarities to members of the β-PFT superfamily known to form channels in targeted membranes. To investigate its mechanism of action, we produced a recombinant protein flanked by an N-terminal 6 histidine tag. Then, we investigated the ability of Biomphalysin to interact with the sporocyst tegument. This optimized protocol describes an ...
[摘要] 血吸虫病或鼠伤寒沙菌是由感染约2亿人的蠕虫属引起的热带病。 寄生虫的生命周期需要作为中间体的淡水蜗牛的特定属的生物疟原虫。 使用采用em的interactome方法。 glabrata plasma和 我们鉴定了一种称为生物溶解素的新的溶细胞蛋白，其显示与已知在靶膜中形成通道的β-PFT超家族成员的相似性。 为了调查其作用机制，我们产生侧翼有N端6组氨酸标签的重组蛋白。 然后，我们调查了Biomphalysin与孢子囊体积相互作用的能力。 这个优化的协议描述免疫细胞化学程序检测组氨酸标记的重组蛋白在孢子被膜上。
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| Cell Proliferation Assay by Flow Cytometry (BrdU and PI Staining)
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Author:
Date:
2012-04-05
[Abstract] Cell Proliferation assays include an important set of fluorescence-based tests that can monitor cell health and cell division by evaluating DNA synthesis through thymidine incorporation. Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a synthetic nucleoside that is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues. BrdU can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle), substituting for thymidine during DNA replication. Antibodies specific for BrdU can then be used to detect the incorporated chemical, thus indicating cells that were actively replicating their DNA. Binding of the antibody requires denaturation of the DNA, usually by exposing the cells to acid or heat. ...
[摘要] 细胞增殖测定包括一组重要的基于荧光的测试，可通过胸苷掺入评估DNA合成来监测细胞健康和细胞分裂。 溴脱氧尿苷（5-溴-2'-脱氧尿苷，BrdU）是是胸苷类似物的合成核苷。 BrdU通常用于检测活组织中的增殖细胞。 BrdU可以掺入到复制细胞的新合成的DNA中（在细胞周期的S期期间），在DNA复制期间替代胸苷。 然后可以使用BrdU特异性的抗体来检测掺入的化学物质，从而指示活跃地复制其DNA的细胞。 抗体的结合需要DNA变性，通常通过将细胞暴露于酸或热。 通常通过用缀合物抗BrdU抗体和DNA染料碘化丙啶（PI）标记以进行细胞周期分析，在流式细胞术中分析BrdU的掺入。
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| Cell Cycle Analysis Using Propidium Iodide Staining with GFP Detection
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Author:
Date:
2012-04-05
[Abstract] Infecting mammalian cells with a GFP construct to overexpress or knockdown target genes is one of the most commonly used methods to study and manipulate gene expression. To determine the target gene function on the cell cycle, analyzing the cell cycle (propidium iodide, PI staining) of GFP positive cells vs GFP negative cells is needed. Usually simple fixation of cells with 70% EtOH for PI staining tends to quench GFP signal; paraformaldehyde (PFA) fixation before ETOH fixation could help to sustain the GFP signal.
[摘要] 用GFP构建体感染哺乳动物细胞以过表达或敲低靶基因是研究和操作基因表达的最常用的方法之一。 为了确定细胞周期上的靶基因功能，需要分析GFP阳性细胞与GFP阴性细胞的细胞周期（碘化丙锭，PI染色）。 通常用70％EtOH的细胞的简单固定用于PI染色倾向于淬灭GFP信号; 多聚甲醛（PFA）固定在ETOH固定之前可能有助于维持GFP信号。
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