| A Phosphopeptide Purification Protocol for the Moss Physcomitrella paten
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Author:
Date:
2015-07-20
[Abstract] Protein phosphorylation is one of the most common post-translational modifications in eukaryotic cells and plays a critical role in a vast array of cellular processes. Efficient methods of protein extraction and phosphopeptide purification are required to ensure the detection of high quality of proteins. In our hands, phenol extraction of proteins and TiO2 chromatography enrich phosphorylated peptides more efficiently than other methods in the moss Physcomitrlla patens (P. patens).
[摘要] 蛋白磷酸化是真核细胞中最常见的翻译后修饰之一，并在大量细胞过程中起关键作用。 需要蛋白质提取和磷酸肽纯化的有效方法以确保高质量的蛋白质的检测。 在我们的手中，蛋白质和TiO 2层析的酚提取比在小麦Physcomitrlla patens中的其它方法更有效地富集磷酸化肽。 ）。
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| In vitro Transcription (IVT) and tRNA Binding Assay
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Author:
Date:
2014-09-20
[Abstract] This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the binding assay to mimic the “natural” situation in the bacterial cell, where the pre-formed, stable tRNA is already present while the T-box leader RNA is actively transcribed by the RNA polymerase. The ...
[摘要] 该方案描述了（i）"活"体外RNA转录与（ii）通过放射性标记的预先形成的tRNA的结合，然后是天然凝胶电泳和磷光成像仪扫描以显现复合物的偶联。 必要性来自一种RNA在不存在其相互作用配偶体时形成的稳定结构。 T盒前导RNA，转录控制系统，折叠成热力学非常稳定的茎 - 环结构，没有tRNA存在，这使得体外结合两个预先形成的RNA的相互作用非常困难。 因此，我调整结合测定以模拟细菌细胞中的"天然"情况，其中预先形成的稳定的tRNA已经存在，而T盒前导RNA被RNA聚合酶主动转录。 方案的第一部分还描述了体外转录和tRNA的标记。
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| Northern Blot of tRNA in Yeast
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Author:
Date:
2013-04-05
[Abstract] tRNAs are small RNAs around 70-90 nt. tRNAs are different from many other small RNAs in that they are very abundant, which makes it difficult to study their transcriptional regulation by traditional northern blot. Traditional Northern blot involves incorporation of radioactive nucleotides through polymerization, however, tRNA is too short for polymerization. Traditional Northern blot detects changes in RNA levels, however, tRNA are so abundant that small changes in their levels will escape detection. For these reasons, metabolic labeling by radioactive Uracil has been used instead. However, metabolic labeling can only examine changes in total tRNA, but cannot distinguish different types of tRNAs. The following protocol describes a method to examine individual tRNA gene transcription by ...
[摘要] tRNA是约70-90nt的小RNA。 tRNA与许多其他小RNA不同，因为它们是非常丰富的，这使得难以通过常规Northern印迹研究它们的转录调节。 传统的Northern印迹涉及通过聚合掺入放射性核苷酸，然而，tRNA对于聚合来说太短。 传统的Northern印迹检测RNA水平的变化，然而，tRNA是如此丰富，其水平的小变化将逃脱检测。 由于这些原因，已经使用了放射性尿嘧啶的代谢标记。 然而，代谢标记只能检查总tRNA的变化，但不能区分不同类型的tRNA。 以下方案描述了通过Northern印迹检查单个tRNA基因转录的方法。
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