| A Quantitative Assay to Measure Stress Granule Association of Proteins and Peptides in Semi-permeabilized Human Cells
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Author:
Date:
2020-12-20
[Abstract] Stress granules (SGs) are membrane-less organelles that form in the cytoplasm through phase separation, in response to diverse stressors. SGs contain translationally stalled mRNAs, proteins involved in translation, and various RNA-binding proteins (RBPs). Due to the high local concentration of aggregation-prone RBPs, SGs might act as condensation sites for aberrant phase transitions of RBPs and could favor formation of solid protein aggregates underlying the pathological cytoplasmic inclusions found in numerous neurodegenerative diseases. Most assays aiming at studying the recruitment of RBPs into SGs are based on overexpression and SG recruitment of RBPs in intact cells. These approaches are, however, often limited by the predominantly nuclear localization of many RBPs, which precludes ...
[摘要] [摘要]圣RESS颗粒剂（SGS）是膜较少细胞器形式在通过相分离细胞质中，响应于不同的压力源。SG包含翻译停滞的mRNA，翻译中涉及的蛋白和各种RNA结合蛋白（RBP）。由于易于凝集的RBP的局部浓度很高，SG可能充当RBP异常相变的缩合位点，并且可能有助于形成在许多神经退行性疾病中发现的病理性细胞质内含物的固体蛋白质聚集体。大多数分析瞄准荷兰国际集团在研究限制性商业惯例的招募SG的基础上表达和SG招聘完整细胞的限制性商业惯例。但是，这些方法通常受到许多RBP的主要核定位的限制，这排除了足以进行SG定位的细胞质RBP浓度，并且没有解决独立于SG形成的RBP募集问题。在这里，我们提出了一个定量的方法来评估重组限制性商业惯例的募集至预先形成的SG，独立于RBP的核定位，用半透细胞和荧光显微镜。在该测定中，SG被第一LY由应激诱导的，然后第质膜ë应激的细胞随后被选择性地透化，以提供重组蛋白的访问的SG。通过用小麦胚芽凝集素阻塞核孔来阻止目的蛋白的核输入。这种测定方法使人们能够定量研究在没有核导入且在受控条件下将RBP募集到SG中的分子机制。该方法允许对野生型，突变体或直接比较翻译后修饰的限制性商业惯例，用于解决第其它蛋白质电子影响“预防或促进限制性商业惯例的SG关联，并且也适用于合成的肽。
图形摘要：
工作流概述在半SG招募重组蛋白质或肽的分析透化细胞 ...
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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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