用于叶片组织亚细胞区室代谢物分析的非水相分级分离法
Non-aqueous Fractionation (NAF) for Metabolite Analysis in Subcellular Compartments of Leaf Tissues.
作者信息
Medeiros David B, Arrivault Stéphanie, Alpers Jessica, Fernie Alisdair R, Aarabi Fayezeh
机构信息
Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476, Potsdam-Golm, Germany.
出版信息
Bio Protoc. 2019 Oct 20;9(20):e3399. doi: 10.21769/BioProtoc.3399.
The accurate determination of metabolite distribution in subcellular compartments is still challenging in plant science. Various methodologies, such as fluorescence resonance energy transfer-based technology, nuclear magnetic resonance spectroscopy and protoplast fractionation allow the study of metabolite compartmentation. However, large changes in metabolite levels occur during such procedures. Therefore, the non-aqueous fractionation (NAF) technique is currently the best method for the study of metabolite distribution. Our protocol presents a detailed workflow including the NAF procedure and quantification of compartment-specific markers for three subcellular compartments: ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker, and nitrate and acid invertase as vacuolar markers.
在植物科学中，准确测定亚细胞区室中的代谢物分布仍然具有挑战性。各种方法，如基于荧光共振能量转移的技术、核磁共振光谱和原生质体分级分离，都可以用于研究代谢物的区室化。然而，在这些过程中代谢物水平会发生很大变化。因此，非水相分级分离（NAF）技术是目前研究代谢物分布的最佳方法。我们的方案提供了一个详细的工作流程，包括NAF程序以及对三个亚细胞区室中特定区室标记物的定量：作为质体标记物的ADP葡萄糖焦磷酸化酶（AGPase）、作为胞质标记物的磷酸烯醇式丙酮酸羧化酶（PEPC），以及作为液泡标记物的硝酸盐和酸性转化酶。
Zotero 插件