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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Author:
Date:
2017-07-05
[Abstract] Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal ...
[摘要] Cdk5活性受两种激活蛋白p35和p39(Tsai et al。,1994; Zheng et al。,1998; Humbert等人)的量的调节,2000)。 p35-Cdk5和p39-Cdk5复合物对盐和洗涤剂浓度的敏感性不同(Hisanaga和Saito,2003; Sato et al。,2007; Yamada等人, 2007; Asada 等人,2008)。 Cdk5激活可以通过Cdk5与其结合的激活剂的免疫沉淀直接测量,随后进行Cdk5激酶测定。在该方案中,用于细胞裂解和免疫沉淀的缓冲液旨在保持p35-和p39-Cdk5复合物以评估总Cdk5活性。裂解细胞,并在核后上清液中测定蛋白浓度。 Cdk5在实验组之间从等量的总蛋白免疫沉淀。然后进行洗涤以除去外来蛋白质并平衡激酶缓冲液中的Cdk5-活化剂复合物。然后将Cdk5与组蛋白H1孵育,组蛋白H1是Cdk5和[γ- 32 P] ATP在体外成功建立的靶标。反应通过SDS-PAGE解析并转移到膜上,用于可视化H1磷酸化和免疫沉淀的Cdk5水平的免疫印迹。我们已经使用该测定来建立p39作为少突神经胶质谱系中Cdk5的主要活化剂。然而,该测定法适用于对裂解条件进行适当调整的其它细胞谱系或组织。 【背景】虽然Cdk5通常与神经元功能相关,但最近的工作已经证明Cdk5也可以调节少突胶质细胞祖细胞(OPC)的发育(Tang等人,1998; ...
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