通过核显微注射来评估异源表达蛋白如何影响非洲爪蟾卵母细胞中的钙离子信号。
Nuclear microinjection to assess how heterologously expressed proteins impact Ca2+ signals in Xenopus oocytes.
作者信息
Lin-Moshier Yaping, Marchant Jonathan S
机构信息
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
出版信息
Cold Spring Harb Protoc. 2013 Mar 1;2013(3):10.1101/pdb.prot072785 pdb.prot072785. doi: 10.1101/pdb.prot072785.
Abstract
The Xenopus oocyte is frequently used for heterologous expression and for studying the spatiotemporal patterning of Ca(2+) signals. Here, we outline a protocol for nuclear microinjection of the Xenopus oocyte for the purpose of studying how subsequently expressed proteins impact intracellular Ca(2+) signals evoked by inositol trisphosphate (InsP3). Injected oocytes can easily be identified by reporter technologies and the impact of heterologously expressed proteins on the generation and properties of InsP3-evoked Ca(2+) signals can be resolved using caged InsP3 and fluorescent Ca(2+) indicators.
摘要
非洲爪蟾卵母细胞常用于异源表达以及研究Ca(2+)信号的时空模式。在此，我们概述了一种用于非洲爪蟾卵母细胞核显微注射的方案，目的是研究随后表达的蛋白质如何影响由三磷酸肌醇（InsP3）诱发的细胞内Ca(2+)信号。通过报告技术可以轻松识别注射后的卵母细胞，并且使用笼锁三磷酸肌醇（caged InsP3）和荧光Ca(2+)指示剂可以解析异源表达蛋白质对InsP3诱发的Ca(2+)信号的产生及特性的影响。
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