| Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
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Author:
Date:
2020-12-05
[Abstract] DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use ...
[摘要] [摘要] DNA足迹是研究蛋白质-DNA相互作用的经典技术。但是，如果蛋白质与DNA的结合较弱，蛋白质的脱落速率较快，或者形成了几种不同的蛋白质-DNA复合物，则传统的足迹方案可能会失败或难以解释。我们的协议不同于传统的足迹协议，因为它提供了一种在使用足迹剂处理后从天然凝胶中分离蛋白质-DNA复合物的方法，从而从游离DNA或其他蛋白质-DNA复合物中去除了结合的DNA。然后从分离的复合物中提取DNA，然后在测序凝胶上电泳以确定印迹模式。该分析为无法使用传统足迹法确定蛋白质与DNA接触的人提供了可能的解决方案。
[背景]核酸酶/化学足迹是一个典型的方法来探测蛋白质-DNA相互作用（腊士和施米茨，1978;萨瑟-德怀特和Gralla，1989;汉普等人，2007） ...
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| Preparation of Yeast tRNA Sample for NMR Spectroscopy
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Author:
Date:
2020-06-20
[Abstract] Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological importance of post-transcriptional RNA modifications is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. By following the ...
[摘要] [摘要 ] 转移RNA（tRNA ）在其生物合成过程中大量修饰有转录后修饰。为了在细胞内履行其功能，tRNA 经历了严格控制的生物生成过程，导致了成熟的tRNA 的形成。此外，tRNA的功能通常是虽然转录后修饰RNA的生物学重要性被广泛理解，方法直接检测它们的RNA生物合成过程中引入是罕见的，并且不容易提供上events.To的时间特性信息获取的信息的tRNA 成熟 在此过程中，我们开发了一种方法，使用NMR作为监测细胞提取物中tRNA 成熟的无中断和连续方式。通过模型酵母tRNA 的时间分辨NMR 成熟，我们发现修饰是该方法的实施需要对具有不同修饰状态的tRNA 样品进行NMR光谱学分析，以鉴定各个修饰的NMR特征。此处将介绍用于NMR光谱分析修饰途径的tRNA 样品的生产，并在酵母tRNA Phe 上进行例证，但可以通过更改构建体的序列扩展到其他tRNA 。该方案描述了未修饰的生产通过体外转录获得tRNA 样品，并通过在大肠杆菌中重组表达tRNA 产生修饰的tRNA 样品。大肠杆菌。
[背景 ] 在生活的各个领域，合成和RNA的成熟包括在特定地点的核苷酸的转录后的化学修饰。在不同的RNA家族，tRNA基因不仅显示最高多种化学修饰，而且密度最高每转录修饰（〜中经修饰的核苷酸8-25％的tRNA 各种生物体的）（Boccaletto ...
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| Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae
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Author:
Date:
2020-03-05
[Abstract] Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell.
[摘要] [摘要 ] 超氧化物歧化酶（SOD能）充当主防御针对反应性氧物质（ROS）通过转换的超氧阴离子自由基（O 2 - ）为分子氧（O 2 ）和过氧化氢（H 2 ? 2 ）。这种酶的家庭成员包括CuZnSODs ，MnSODs ，FeSODs 和NiSODs ，这取决于是需要适当的活动辅助因子的性质。大多数真核生物，包括酵母，都具有CuZnSOD 和MnSOD 。该协议旨在评估酵母的活性 使用硝基蓝四唑染色法从细胞提取物中提取酿酒酵母MnSOD Sod2p 。该方法可用于估计Mn 2+ 的细胞生物利用度以及评估细胞的氧化还原状态。
[背景 ] 的SODs被定义为减少正常有氧代谢为氧气和过氧化氢期间形成的氧的有害自由基含金属的抗氧化剂酶。：这些酶是基于需要作为辅因子进行适当的酶活性的金属分类CuZnSODs ，MnSODs ，FeSODs ，和NiSODs 。在酿酒酵母中，有两个S OD ：CuZn-Sod1p和Mn-Sod2p（Abreu和Cabelli ...
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