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30 TUBE

30 TUBE

Company: Krackeler Scientific
Catalog#: 6-45500-30-CS
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Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts
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2017-01-05
[Abstract]  Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [45Ca] and [54Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [45Ca] or [54 ... [摘要]  测定Ca 2 + 和Mn 2 + 的相对分布是分析在叶绿体包膜或类囊体中钙和/或锰转运蛋白水平发生变化的突变体的重要工具膜。该方案中描述的方法允许使用同位素[ 45 Ca]和[ 54 Mn]定量分析叶绿体基质和类囊体之间钙和锰离子的相对分布,作为放射性示踪剂。为了避免非叶绿体膜系统的污染,该方法被设计用于分离拟南芥的纯和完整的叶绿体。通过Percoll梯度离心分离完整的叶绿体。然后在轻孵育步骤期间使叶绿体吸收[ 45 Ca]或[ 54 Mn]。孵化后,叶绿体保持完整或渗透/机械处理以释放类囊体。通过液体闪烁计数可以确定掺入量[ 45 Ca]或[ 54 Mn]的量,并计算相对分布。

背景 钙和锰是光系统II的结构组分,形成无机Mn 4 O 5 O 5 O 3簇,其中水氧化发生电子,质子和分子氧的结果。通过叶绿体包膜和类囊体膜的Ca 2+ / +和/或超氧化物质是使得植物细胞满足PSII对这些阳离子的高需求的基本过程。在先前的研究中,使用来自豌豆植物的分离的类囊体膜(Ettinger等人)分析了Ca 2 + / H ...

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