| 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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| Analysis of L- and D-Amino Acids Using UPLC
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Author:
Date:
2014-09-05
[Abstract] With the exception of glycine, α-amino acids are optically active, and two optical isomers (L- and D-) of each amino acid can be formed. Recent developments of analytical techniques have revealed that several free D-amino acids such as D-aspartate, D-serine and D-alanine exist in many kinds of organism including human and have biologically important roles. D-Aspartate regulates reproductive activity in animals and humans. D-Serine serves as a co-agonist of the N-methyl-D-aspartate receptor, which mediates glutamatergic neurotransmission. D-Alanine plays a role like osmolyte in crustaceans and mollusks. In this protocol, we describe a method for analysis of L- and D-amino acids using ultra-performance liquid chromatography (UPLC). To analyze D- and L-amino acids, the enantiomers ...
[摘要] 除了甘氨酸，α-氨基酸具有光学活性，并且可以形成每个氨基酸的两种光学异构体（L-和D-）。分析技术的最新发展表明，几种游离D-氨基酸如D-天冬氨酸，D-丝氨酸和D-丙氨酸存在于包括人在内的许多种生物中，并且具有生物学上的重要作用。 D-天冬氨酸调节动物和人的生殖活性。 D-丝氨酸作为Nε-甲基-D-天冬氨酸受体的共激动剂，其介导谷氨酸能神经传递。 D-丙氨酸在甲壳类动物和软体动物中起到类似渗透物的作用。在该协议中，我们描述了使用超高效液相色谱（UPLC）分析L-和D-氨基酸的方法。为了分析D-和L-氨基酸，首先将对映异构体转化为非对映异构体（非对映异构体是与物镜和镜像无关的立体异构体，并且不是对映异构体），使用前柱衍生化，邻苯二甲醛加上Nε - 酰化的半胱氨酸（Nε - 乙酰基-L-半胱氨酸或Nε - 叔丁基氧羰基-L 。所得衍生物是荧光非对映异构体。然后使用UPLC在十八烷基硅烷基固定相上分离所得荧光异吲哚衍生物，并通过包括在UPLC系统中的荧光检测器检测荧光。使用该方法，可以分析16种D-氨基酸。
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