| Analysis of Isotopically-labeled Monogalactosyldiacylglycerol Molecular Species from [14C]Acetate-Labeled Tobacco Leaves
|
|
Author:
Date:
2020-12-20
[Abstract] Plant lipid metabolism is a dynamic network where synthesis of essential membrane lipids overlaps with synthesis of valuable storage lipids (e.g., vegetable oils). Monogalactosyldiacylglycerol (MGDG) is a key component of the chloroplast membrane system required for photosynthesis and is produced by multiple pathways within the lipid metabolic network. The bioengineering of plants to enhance oil production can alter lipid metabolism in unexpected ways which may not be apparent by static quantification of lipids, but changes to lipid metabolic flux can be traced with isotopic labeling commonly with [14C]acetate. Because lipid classes such as MGDG are composed of many different molecular species, full analysis of metabolically labeled lipids requires separation and quantification of the ...
[摘要] [摘要]植物脂质代谢是一个动态的网络，其中与宝贵的存储脂质（合成所必需的膜脂质重叠合成例如，植物油）。单半乳糖基二酰基甘油（MGDG）是光合作用所需的叶绿体膜系统的关键组成部分，由脂质代谢网络内的多种途径产生。通过植物的生物工程来提高石油产量，可以以意想不到的方式改变脂质的代谢，而脂质的静态定量检测可能无法察觉到这种变化，但是通常可以通过同位素标记[ 14 C]乙酸盐追踪脂质代谢通量的变化。由于脂类（例如MGDG）由许多不同的分子种类组成，因此对代谢标记的脂类进行全面分析需要分离和定量单个标记的分子种类，而这通常是通过薄层色谱法进行的。在这里，我们提出了一种反相HPLC方法，用于在35分钟内从烟叶中分离MGDG分子种类。每个14 C标记的分子种类的定量通过在线流动无线电检测器完成。这种通过放射HPLC分析[ 14 C]乙酸标记的MGDG分子种类的方法提供了一种快速，高通量和可靠的分析方法，以鉴定由于生物工程或其他代谢紊乱引起的MGDG代谢变化。
背景] Monogalactosyldiacylglycerol （MGDG）是一类的甘油脂两种脂肪酸链，其包括与附连到半乳糖部分SN ...
|
|
|
| Buoyant Density Fractionation of Small Extracellular Vesicle Sub-populations Derived from Mammalian Cells
|
|
Author:
Date:
2020-08-05
[Abstract] Small extracellular vesicles (sEVs) encompass a variety of distinct vesicles that are secreted to the extracellular space. Many methodologies currently used for EV isolation (e.g., differential ultracentrifugation concluding in a high-speed pellet, precipitation by macromolecular crowding agents or size excusion chromatography–SEC) do not fractionate distinct sEV sub-populations. Samples obtained by the aforementioned methods are usually used for characterization and physiological studies. However the fraction that contains the molecule of interest or is the carrier of a specific activity is unknown. Therefore isolating distinct sEV sub-populations is critical to understand EV function. The goal of this procedure is to purify distinct sEV sub-populations based on slight ...
[摘要] [摘要] 小细胞外小泡（sEVs）包括分泌到细胞外空间的各种不同的小泡。目前用于EV分离的许多方法（例如，高速颗粒中的差速超速离心、大分子拥挤剂沉淀或尺寸排除色谱法）没有分离不同的sEV亚群。通过上述方法获得的样品通常用于表征和生理学研究。然而，包含感兴趣分子或特定活性载体的部分是未知的。因此，分离不同的sEV亚群对于理解EV功能至关重要。该程序的目的是基于它们浮力密度的微小差异来纯化不同的sEV亚群。此外，该技术还允许从高速颗粒中共同分离的无囊泡RNA蛋白复合物中或通过使用拥挤剂来纯化sEVs。该方案描述了用于收集sEV的哺乳动物细胞的培养、sEV沉淀、sEV亚群的浮力密度分馏和sEV标记的免疫印迹。该方法可用于分离由多种哺乳动物细胞产生的不同的sEV亚群。
[] ...
|
|
|