| 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 ...
[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 ...
[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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