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UltraPureTM乙二胺四乙酸,二钠盐,二水合物

Company: Thermo Fisher Scientific
Catalog#: 15576028
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Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy
Author:
Date:
2021-03-05
[Abstract]  

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to

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[摘要]  [摘要]在细菌中,停滞的DNA复制叉的重新启动需要DN A解旋酶PriA 。PriA可以识别并重塑废弃的DNA复制叉,在3'到5'方向展开DNA,并促进解旋酶DnaB加载到DNA上以重新开始复制​​。ssDNA结合蛋白(SSB)通常存在于废弃的叉子上,从而保护ssDNA免受核酸酶的破坏。该研究是基于所述测定法离解结,表面等离振子共振,单分子FRET,和x射线晶体结构已经揭示的解旋酶活性PRIA ,SSB- PriA相互作用以及PriA解旋酶的结构信息。在这里,我们使用原子力显微镜(AFM)可视化了在不存在ATP的情况下在有或没有SSB的情况下PriA和带有或不带有SSB的DNA底物之间的相互作用,以描绘PriA在其ATP催化的DNA解链反应之前的底物识别模式。该协议描述了获取高分辨率AFM图像的步骤以及数据分析和表示的细节。

[背景]当DNA复制遇到障碍或断裂时,需要对其进行修复并随后重新启动(Kogoma,1997; Cox等,2000; McGlynn和Lloyd,2002;G abbai和Marians,2010; Michel等,2018)。 )。在细菌中,DNA解旋酶PRIA通过识别废弃DNA复制叉,从而便于重新组装的介导这一过程复制体的解旋酶和装载DNAB (Wickner和赫维茨,1975; Zavitz和Marians,1992; ...

Cell Synchronization by Double Thymidine Block
Author:
Date:
2018-09-05
[Abstract]  Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at G1/S boundary by using double thymidine block. After release into normal medium, cell population at distinct cell cycle phase could be collected at different time points. [摘要]  细胞同步广泛用于研究涉及细胞周期进程调节的机制。 通过同步,可以获得不同细胞周期阶段的细胞。 胸苷是一种DNA合成抑制剂,可在DNA复制前阻止细胞在G1 / S边界。 在这里,我们提出了使用双胸苷阻滞同步G1 / S边界细胞的协议。 释放到正常培养基中后,可以在不同的时间点收集不同细胞周期阶段的细胞群。

【背景】细胞周期和细胞分裂是细胞生物学的核心。为了构建多细胞生物体,细胞复制对于产生可以执行特定功能的特化细胞是必需的。正常细胞周期由间期(G1期,S期和G2期)和有丝分裂期(M期)组成(Rodríguez-Ubreva et al。,2010;Léger et al。 ,2016)。在间期期间,遗传物质被复制并使一切为有丝分裂做好准备。然而,在有丝分裂期,重复的染色体被分离并分配到子细胞中(Sakaue-Sawano 等人,,2008)。

为了精确保存遗传信息,必须严格控制细胞周期进程。细胞周期蛋白/ CDK复合物通过在各自阶段快速促进活动来控制细胞周期进展,并且当它们的阶段完成时迅速失活(Graña和Reddy,1995)。

细胞同步对于研究细胞周期调节事件特别有用。使用不同的方法,细胞可以在不同的细胞周期阶段同步。治疗作为微管形成抑制剂的诺考达唑可以使细胞处于G2 / M期同步(Ho et ...

Whole-mount Enteroid Proliferation Staining
Author:
Date:
2016-06-20
[Abstract]  Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology in vitro as they recapitulate the various epithelial cells within the intestinal crypt (Mahe et al., 2013; Sato et al., 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations in gene expression, treatment with protective and toxic agents, and genetic mutations alter properties essential for crypt growth and survival as well as the stem cell properties of the individual cells within the crypt. This protocol describes a method of visualization of proliferating cells within the crypt in three dimensions (Barrett et al., 2015). Whole-mount proliferation staining of enteroids ... [摘要]  小肠类器官,也称为肠袢,已经成为越来越多地用于肠道生物学的体外模型,因为它们重现了在肠隐窝内的各种上皮细胞(Mahe等人 ,2013; Sato et al。,2009)。在这些培养物中评估生长动力学是理解基因表达的改变,用保护性和毒性试剂处理以及遗传突变改变隐窝生长和存活所必需的性质以及细胞内单个细胞的干细胞性质的重要步骤地穴。该方案描述了在隐窝内三维的增殖细胞的可视化方法(Barrett等人,2015)。使用EdU掺入的肠衣的整体增殖染色使得研究者能够观察到肠内的所有增殖细胞,而不是如在包埋和切片中所见到的在薄切片中获得生长信息,确保来自干细胞区室的增殖的真实表现到隐窝的终末分化细胞。

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