| Measurement of PI4P Levels in Intact Chloroplasts Isolated from Arabidopsis thaliana
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Author:
Date:
2016-02-05
[Abstract] Phosphatidylinositol 4-phosphate (PI4P), a major species of phosphoinositides, modulates many fundamental cellular processes. We have recently revealed that PI4P plays an important role in chloroplast division as a negative regulator. Despite its importance in chloroplasts, the content of PI4P in chloroplasts is very low and it is difficult to measure PI4P levels. In this protocol, we describe a simple method that we have developed for measurement of low level of PI4P in chloroplasts. Intact chloroplasts were isolated by a basic method using Percoll gradient centrifugation and acidic lipids were extracted from the isolated chloroplasts. The extracted acidic lipids including PI4P were spotted onto the membrane strip, which had been pre-spotted with PI4P standards and other ...
[摘要] 磷脂酰肌醇4-磷酸（PI4P）是磷酸肌醇的主要种类，调节许多基本的细胞过程。 我们最近揭示PI4P在叶绿体分裂作为负调节器中发挥重要作用。 尽管其在叶绿体中的重要性，PI4P在叶绿体中的含量非常低，并且难以测量PI4P水平。 在这个协议，我们描述了一个简单的方法，我们已经开发测量叶绿体中的低水平的PI4P。 通过使用Percoll梯度离心的碱性方法分离完整的叶绿体，并从分离的叶绿体中提取酸性脂质。 将包括PI4P的提取的酸性脂质点样在膜条上，其已经用PI4P标准品和其它磷酸肌醇作为阴性对照预先点样。 使用PI4P结合蛋白检测膜上酸性脂质斑点中的PI4P。
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| Preparation of Chloroplast Lipid Membrane and Lipid-protein Interaction Assay
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Author:
Date:
2016-02-05
[Abstract] Lipid-Protein interaction assay is a method to search lipids, which are bound with proteins in vitro. Since membranes that are spotted with chloroplast lipids such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) are not commercially available, we extracted these lipids from cyanobacterial cells and spotted them onto membranes. The prepared membranes could be used for lipid-protein interaction assay.
[摘要] 脂质 - 蛋白质相互作用测定法是一种搜索在体外与蛋白质结合的脂质的方法。 由于用叶绿体脂质例如单半乳糖基二酰基甘油（MGDG），二半乳糖基二酰基甘油（DGDG）和磺基奎诺糖基二酰基甘油（SQDG）点样的膜不可商购，因此我们从蓝藻细胞中提取这些脂质并将它们点样到膜上。 所制备的膜可用于脂质 - 蛋白质相互作用测定。
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