| Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings
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Author:
Date:
2017-08-05
[Abstract] The plant endomembrane system plays vital roles for synthesis, modification and secretion of proteins and lipids. From the classic view, only mRNAs encoding secreted proteins could be targeted to the endoplasmic reticulum (ER) for translation via a co-translational translocation manner, however, recently this model has been challenged by accumulative evidence that lots of cytosolic mRNAs could also associate with ER, and that some categories of small RNAs are enriched on ER. These results suggested unrevealed functions of ER beyond our current knowledge. The large scale identification of RNAs and proteins on microsome is crucial to demonstrating the ER function and the studies will be boosted by next generation sequencing technology. This protocol provides a technical workflow to isolate ...
[摘要] 植物内膜系统对蛋白质和脂质的合成，修饰和分泌起着至关重要的作用。 从经典观点来看，只有编码分泌蛋白质的mRNA才能通过协同翻译方式靶向内质网（ER）进行翻译，然而最近，这一模型已经被大量的细胞溶质mRNA也可能与 ER，并且一些类别的小RNA在ER上富集。 这些结果表明ER的功能超出了目前的知识。 在微粒体上大规模鉴定RNA和蛋白质对于显示ER功能至关重要，研究将由下一代测序技术提升。 该协议提供了从植物组织中分离细胞质，微粒体，游离多聚体（FP）和膜结合多聚体（MBP）的技术工作流程。 分离的级分适用于mRNA，小RNA和蛋白质的基因组广谱分析。
【背景】植物内膜系统对于细胞壁形成，脂质生物合成，蛋白质合成，修饰，折叠和贩运非常重要。根据共翻译易位模型，分泌蛋白N末端的信号肽由细胞溶质多核糖体合成，然后由ER上的信号识别粒子识别，其余蛋白质部分随后在ER上合成。根据该模型，只有编码分泌蛋白的mRNA可以被带到ER进行翻译（Peter和Johnson，1994）。然而，从哺乳动物和植物细胞ER（Lerner等人，2003; de ...
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| Polysome Fractionation to Analyze mRNA Distribution Profiles
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Author:
Date:
2017-02-05
[Abstract] Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ...
[摘要] 真核细胞通过精确调节特定基因产物的表达来适应外部或内部信号的变化。蛋白质编码基因的表达受到转录和转录后水平的控制。在后面的步骤中，翻译的调节在需要蛋白质表达模式快速变化的细胞过程中特别重要。 mRNA的翻译效率由RNA结合蛋白（RBP）和非编码（nc）RNA如微RNA（Panda等人，2014a和2014b; Abdelmohsen等人）改变2014）。调节选择性mRNA翻译的因素的影响是RNA生物学中的一个关键问题。多核糖体（多核糖体）分馏分析是评估核糖体与给定mRNA的关联的有效方法。它提供了关于该mRNA的翻译状态的有价值的信息，这取决于与它们相关联的核糖体的数目，并且鉴定未翻译的mRNA（Panda等人，2016）。与许多核糖体相关的mRNA形成大量的多核糖体，预计将被主动翻译，而与少数或没有核糖体相关的mRNA有可能翻译不佳。总之，多聚糖分馏分析允许直接测定整个转录组水平的翻译效率以及个体mRNA。
背景 ...
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| Isolate and Sub-fractionate Cell Membranes from Caulobacter crescentus
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Author:
Date:
2012-10-20
[Abstract] Cell membranes from Caulobacter can be isolated and separated into inner and outer membranes according their characteristic buoyant densities on a sucrose gradient. Fractionation can be used to determine the localisation of uncharacterised proteins and to enrich protein complexes present in either of these membranes for biochemical analysis such blue-native PAGE and immunoprecipitation.
[摘要] 来自弯曲杆菌的细胞膜可以根据蔗糖梯度上的特征浮力密度分离并分离成内膜和外膜。 分馏可以用于确定未表征的蛋白质的定位并富集存在于这些膜的任一个中的蛋白质复合物用于生物化学分析，例如蓝色天然PAGE和免疫沉淀。
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