Analysis of Peroxisome Biogenesis by Phos-Tag SDS-PAGE
Analysis of Peroxisome Biogenesis by Phos-Tag SDS-PAGE
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通过 Phos-Tag SDS-PAGE 分析过氧化物酶体生物合成
DOI：
10.1007/978-1-0716-3048-8_15
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发表时间：
2023
影响因子：
--
通讯作者：
Fujiki Yukio
中科院分区：
文献类型：
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作者：
Okumoto Kanji;Fujiki Yukio
Phos-tag是一种选择性的磷酸盐结合分子，基于Phos-tag的方法已被开发用于研究磷蛋白质组。在使用phos标签衍生物的各种分析技术中，磷酸盐亲和电泳使用phos标签丙烯酰胺，称为phos标签SDS-PAGE，可以分离磷酸化蛋白，并从聚丙烯酰胺凝胶中的非磷酸化蛋白中迁移较慢。phos标签SDS-PAGE的程序与传统SDS-PAGE的程序基本相同，因此易于在所有实验室使用。Phos-tag SDS-PAGE被广泛应用于根据磷酸基团的数量和/或位点对整体磷酸化状态进行定量分析。phos标签SDS-PAGE也被引入到过氧化物酶体研究领域，包括氧化应激诱导的和有丝分裂特异性的Pex14磷酸化，Pex14是过氧化物酶体基质蛋白转运机制复合体的核心组成部分。在这里，我们描述了一种实用的phos标签SDS-PAGE方案及其在过氧化物酶体生物发生研究中的应用。
Phos-tag, a selective phosphate-binding molecule, and Phos-tag-based methodologies have been developed to investigate the phosphoproteome. In various analytical techniques using Phos-tag derivatives, phosphate-affinity electrophoresis using Phos-tag acrylamide, called Phos-tag SDS-PAGE, enables separation of phosphorylated proteins with a slower migration from non-phosphorylated proteins in polyacrylamide gels. The procedures for Phos-tag SDS-PAGE are largely common to those for conventional SDS-PAGE, thus being readily available for all laboratories. Phos-tag SDS-PAGE is widely applied to quantitative analysis of the overall phosphorylation state depending on the number and/or sites of the phosphate group. Phos-tag SDS-PAGE has also been introduced to the field of peroxisome study, including oxidative stress-induced and mitosis-specific phosphorylation of Pex14, a central component of the translocation machinery complex for peroxisomal matrix proteins. Here, we describe a practical protocol for Phos-tag SDS-PAGE and its application to peroxisome biogenesis research.