| Expression and Purification of Recombinant Skd3 (Human ClpB) Protein and Tobacco Etch Virus (TEV) Protease from Escherichia coli
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Author:
Date:
2020-12-05
[Abstract] Skd3 (encoded by human CLPB) is a mitochondrial AAA+ protein comprised of an N-terminal ankyrin-repeat domain and a C-terminal HCLR-clade nucleotide-binding domain. The function of Skd3 has long remained unknown due to challenges in purifying the protein to high quality and near homogeneity. Recently we described Skd3 as a human mitochondrial protein disaggregase that solubilizes proteins in the mitochondrial intermembrane space. This protocol overcomes the challenges associated with purifying Skd3 and allows for in depth in vitro study of Skd3 activity. Tobacco etch virus (TEV) protease is required in the purification of Skd3. Thus, we also describe how to purify high quality TEV protease for use in the purification of Skd3, other purification protocols, and in vitro assays requiring TEV ...
[摘要] [摘要] Skd3（由人类CLPB编码）是一种线粒体AAA +蛋白，由N末端锚蛋白重复域和C末端HCLR分支核苷酸结合域组成。由于在纯化蛋白质达到高质量和接近均质性方面的挑战，Skd3的功能长期未知。最近，我们描述Skd3作为人类线粒体蛋白disaggregase ，在线粒体膜间间隙增溶蛋白。该协议克服了与纯化Skd3相关的挑战，并允许对Skd3活性进行深入的体外研究。Skd3的纯化需要烟草蚀刻病毒（TEV）蛋白酶。因此，我们还描述了如何净化高质量TEV蛋白酶可用于纯化Skd3，其他纯化方案以及需要TEV蛋白酶的体外测定。
[背景] Skd3是一种线粒体AAA +蛋白，与多系统线粒体疾病VII型3-甲基谷氨酸酸尿症（MGCA7）有关（Capo-Chichi等人，2015； Kanabus等人，2015； Saunders等人，， 2015; Wortmann等人，2015 ; Kiykim等人，2016 ）。由于在体外研究Skd3的能力有限，因此对生物学功能和这些突变对Skd3活性的影响的研究仍难以捉摸（Cupo和Shorter，2020； ...
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| Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations
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Author:
Date:
2020-07-05
[Abstract] Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal reproducibility and low sample success rate as accepted caveats. However, low sample success rates are an obvious disadvantage when polytene chromosomes are used for more high-throughput approaches, such as genetic or antibody screens, or for experiments requiring high-quality chromosome structure preservation. Here we present an exceptionally reproducible squashing and fluorescence staining protocol, which generates high-quality fluorescence images on ...
[摘要] [摘要] 果蝇几十年来，幼虫唾液腺多线染色体压片被用来分析全基因组蛋白质结合模式、转录激活过程以及特定基因位点染色质结构的变化。在过去的几年中，压榨方案已经有了许多改进，次优的重复性和较低的样本成功率是公认的警告。然而，当多线染色体用于更高通量的方法，如遗传或抗体筛查，或用于需要高质量染色体结构保存的实验时，低样本成功率是一个明显的缺点。在这里，我们提出了一个特别可重复的挤压和荧光染色协议，它可以在扩散良好的染色体上生成高质量的荧光图像。接下来是我们的新的、半自动的MATLAB分析程序，用于以逐像素的方式确定多线染色体上单个位点上感兴趣的荧光信号之间的相关性。在我们的案例中，我们已经用这种方法来评估染色体在基因组位点的变化，这些位点被核孔蛋白的异位靶向。我们的分析程序的使用提高了对染色质结构的变化或染色质的蛋白质补充的无偏结论的能力，而不考虑免疫荧光染色的样本变化。由于它只是基于特定位置的荧光强度的差异，因此所提供的分析程序不局限于对多线染色体的分析，并且可以应用于许多不同的上下文中，其中任何特定位置的荧光信号之间的相关性是感兴趣的。
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| Live Cell Measurement of the Intracellular pH of Yeast by Flow Cytometry Using a Genetically-Encoded Fluorescent Reporter
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Author:
Date:
2020-06-20
[Abstract] The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to measure intracellular pH. While microscopy is often used to make such measurements, it is relatively low-throughput such that collecting enough data to fully characterize populations of cells is challenging. Flow cytometry avoids this drawback, and is a powerful tool that allows for rapid, high-throughput measurement of fluorescent readouts in individual cells. When combined with pH-sensitive fluorescent reporters, it can be used to characterize the intracellular pH of large populations of cells at the single-cell level. We adapted microscopy and ...
[摘要] [摘要 ] 酵母的细胞内PH是对生长状态和环境条件响应发生变化的严格调节的生理线索。荧光报告基因可响应局部PH的变化而改变荧光，可用于测量细胞内的PH。常被用来进行此类测量，它是相对低吞吐量，从而收集足够的数据来充分体现群体的细胞是一种挑战。˚F 低流式细胞术避免了这种弊病，而且是一个强大的工具，允许对于快速，高通量测量单个细胞中的荧光读数。 当与pH敏感荧光报告相结合，它可以被用来表征的细胞内pH 大人口小号在单细胞的细胞level.We适于显微镜和流式细胞术为基础的方法来测量酵母胞内pH值。细胞可以可以在接近自然的条件下生长直到测量点为止，并且该协议可以在变化的环境条件下适用于单点或动态（时间分辨）测量。
[背景 ] 酵母的细胞内pH值与像活力和生长速率特性相关，和细胞内pH的调节消耗的相当大的比例的细胞活力的资源（Orij 等人，2011） 。然而，细胞内pH值可迅速变化并且是高度对环境敏感，因此对于细胞生理学的这一重要方面，采用快速，最小扰动的测量方法至关重要。
将目标化合物的局部浓度转换为荧光读数的基因编码生物传感器彻底改变了我们表征细胞内环境的能力，对于某些传感器，绝对荧光强度与读数相关，这在细胞内进行时可能是一个问题测量，因为荧光依靠小号都在该传感器的表达水平，而略有差异小区到小区，和特征兴趣。[R ...
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