| Chromatin Fractionation Assay in Fission Yeast
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Author:
Date:
2014-07-20
[Abstract] The protein recruitment onto chromatin is a critical process for DNA metabolism, including DNA replication, DNA repair and DNA recombination. Especially DNA modification enzymes and checkpoint proteins are loaded onto DNA damage sites in a context-dependent manner. In our recent study (Kunoh and Habu, 2014), the chromatin association of Pcf1, a large subunit of Chromatin Assembly Factor-1 (CAF-1), was monitored after exposure of cells to hydroxyurea which slowed down the DNA replication. Results of the chromatin fractionation assay provided evidence that Pcf1 was recruited to chromatin upon DNA replication stress. A similar procedure enabled to reveal the chromatin association of Orp1, Mcm proteins, and Swi6 (Sadaie et al., 2008; Ogawa et al., 1999). This assay allows us ...
[摘要] 染色质上的蛋白质募集是DNA代谢的关键过程，包括DNA复制，DNA修复和DNA重组。特别是DNA修饰酶和检查点蛋白以上下文依赖性方式装载到DNA损伤位点。在我们最近的研究中（Kunoh和Habu，2014），在将细胞暴露于羟基脲（其减慢DNA复制）后，监测Pcf1的染色质缔合，其为染色质装配因子-1（CAF-1）的大亚基。染色质分馏测定的结果提供了Pcf1在DNA复制应激时募集到染色质的证据。类似的程序能够揭示Orp1，Mcm蛋白和Swi6的染色质缔合（Sadaie等人，2008; Ogawa等人，1999）。该测定允许我们从活细胞分离染色质结合和非结合蛋白。以下各部分的免疫印迹提供了关于我们的靶蛋白的染色质结合状态的信息。
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| Preparation of Parasite Protein Extracts and Western Blot Analysis
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Author:
Date:
2014-06-05
[Abstract] In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available to achieve this. If the protein is present within the parasite or is attached to a cellular structure of the iRBC cell, saponin can be used. This reagent lyses the membranes of infected and uninfected erythrocytes, the Maurer´s clefts (vesicular structures in the iRBC) and the parasitophorous vacuole membrane containing the parasite but leaves the parasite plasma membrane intact, providing a convenient procedure to isolate intact parasites ...
[摘要] 为了制备用于蛋白质印迹分析的恶性疟原虫血液阶段的蛋白质提取物，需要将感染的红细胞（iRBC）与构成寄生虫的主体的未感染的红细胞（uRBC）分离文化。根据感兴趣的寄生虫蛋白的定位，可以使用不同的方法来实现这一点。如果蛋白质存在于寄生虫内或附着于iRBC细胞的细胞结构，则可以使用皂苷。该试剂裂解感染的和未感染的红细胞的膜，Maurer's裂口（iRBC中的囊泡结构）和含有寄生虫的寄生虫液泡膜，但是使寄生虫质膜完整，提供了一种方便的方法来分离没有uRBC的完整寄生虫。然而，该方法的缺点是iRBC的宿主细胞胞质和寄生泡（PV）含量丢失。如果这必须避免，可以使用Percoll梯度将完整的iRBC与uRBC分离。然后可以使用Tetanolysin和皂苷的顺序处理来从寄生虫选择性释放iRBC细胞溶质和PV含量。这些选择性裂解方法也适合于确定目标蛋白质的亚细胞定位
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| Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites
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Author:
Date:
2014-06-05
[Abstract] This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could ...
[摘要] 该方案用于制备过表达用GFP标记的寄生虫蛋白PF13_0191的恶性疟原虫血液阶段寄生虫的预嵌入样品。使用GFP特异性抗体和蛋白A-Gold，使用标准透射电子显微镜（EM）测定过表达的蛋白在感染的宿主细胞中的定位。预嵌入EM是其中在包埋和切片之前引入抗体的常见方法。该方法避免了嵌入后在EM-切片上通常难以检测抗原的问题。在本文所述的方法中，检测寄生虫感染的宿主细胞中的抗原。通过宿主细胞用破伤风溶蛋白透化可以进入抗体。原则上，如果样品在添加相关抗体之前被适当地固定和透化，则该方法也可以用于检测寄生虫内的抗原。虽然抗体的获取将避免嵌入后方法中经常见到的检测问题，但是该方法将产生相对较差的形态。
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