| [14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae
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Author:
Date:
2015-12-20
[Abstract] The gram-negative curved bacillus Vibrio cholerae (V. cholerae) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able to enter the cell. In this protocol, we show how to measure linoleic acid entering V. cholerae when added exogenously and determine whether it is able to enter the cytoplasm. This protocol will quantify how much linoleic acid is able to enter the cell and then identify the amount of linoleic acid that stays in the membrane or ultimately enters the cytoplasm.
[摘要] 革兰氏阴性弯曲杆菌霍乱弧菌（<霍乱弧菌）导致严重的腹泻病霍乱。 这里介绍的工作是评估是否不饱和脂肪酸（ufas），如亚油酸，有可能直接影响参与dna结合的蛋白质，因为他们能够进入细胞。 在这个协议，我们显示如何测量亚麻酸进入。 霍乱菌，并且确定它是否能够进入细胞质。 该方案将量化亚油酸能够进入细胞多少，然后鉴定保留在膜中或最终进入细胞质的亚油酸的量。
这里介绍的工作是评估是否不饱和脂肪酸（ufas），如亚油酸，有可能直接影响参与dna结合的蛋白质，因为他们能够进入细胞。="" 在这个协议，我们显示如何测量亚麻酸进入。="" 霍乱菌，并且确定它是否能够进入细胞质。="" 该方案将量化亚油酸能够进入细胞多少，然后鉴定保留在膜中或最终进入细胞质的亚油酸的量。="">
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| Isolation of Polysome-bound mRNA from Rice Solid Tissues Amenable for RT-PCR and Profiling Experiments
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Author:
Date:
2015-03-05
[Abstract] Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and western blotting, genome-wide microarray analysis or qRT-PCR. Here, we describe a method for the isolation and quantification of high-quality polysome-bound mRNA complexes from small quantities of liquid-nitrogen-frozen solid tissue samples of rice shoots/roots. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level.
[摘要] Polysome谱分析是翻译控制研究中经常执行的任务，不仅能够直接监测翻译的效率，而且可以很容易地扩展到广泛的下游应用，如Northern和western印迹，全基因组微阵列分析或qRT- PCR。 在这里，我们描述了一种从少量液氮冷冻固体组织样本的水稻芽/根中分离和定量高质量多糖结合mRNA复合物的方法。 获得的mRNA可以通过在单个转录物或全局水平评估多核糖体mRNA丰度的方法进一步分析。
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| Polysome Preparation, RNA Isolation and Analysis
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Author:
Date:
2012-11-05
[Abstract] During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosomes can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.
[摘要] 在mRNA翻译期间，40S和60S核糖体亚基结合靶mRNA形成80S复合物（单体）。 这种核糖体在翻译延长过程中沿着mRNA移动以促进tRNA阅读密码子，其中翻译被激活并且许多单体可以同时结合相同的mRNA，其形成多核糖体。 多聚体可以通过蔗糖密度梯度离心进行大小分级。 多核糖体中更特异的mRNA意味着mRNA的更有活性的翻译状态。
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