Identification of myo-inositol-binding proteins by using the biotin pull-down strategy in cultured cells.
Identification of myo-inositol-binding proteins by using the biotin pull-down strategy in cultured cells.
复制标题
利用生物素下拉策略在培养细胞中鉴定肌醇结合蛋白。
DOI：
10.1016/j.xpro.2022.101385
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发表时间：
2022-06-17
期刊：
影响因子：
--
通讯作者：
Lin, Hui-Kuan
中科院分区：
文献类型：
--
作者：
Hsu, Che-Chia;Xu, Zhi-Gang;Lei, Jie;Chen, Zhong-Zhu;Li, Hong-Yu;Lin, Hui-Kuan
代谢物不仅是代谢反应的底物，而且还作为信号分子调节多种生物功能。代谢物结合蛋白的鉴定有助于了解它们在参与的经典代谢途径之外的功能。我们提供了合成生物素标记的肌醇的方案，该方案用于通过使用生物素下拉测定来鉴定其结合蛋白，鉴于没有可用的工具用于在细胞和体外系统中快速筛选肌醇结合蛋白。因此，生物素标记的肌醇探针提供了一种鉴定肌醇传感器的工具。有关本方案使用和执行的完整详细信息，请参阅。生物素标记的肌醇下拉试验的详细方案合成和验证生物素缀合的肌醇部分通过生物素-链霉亲和素方法鉴定肌醇结合蛋白使用未标记的肌醇验证特定的肌醇结合蛋白出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。代谢产物不仅是代谢反应中的底物，而且还作为信号分子调节多种生物学功能。代谢物结合蛋白的鉴定有助于了解它们在参与的经典代谢途径之外的功能。我们提供了合成生物素标记的肌醇的方案，该方案用于通过使用生物素下拉测定来鉴定其结合蛋白，鉴于没有可用的工具用于在细胞和体外系统中快速筛选肌醇结合蛋白。因此，生物素标记的肌醇探针提供了一种鉴定肌醇传感器的工具。
Metabolites are not only substrates in metabolic reactions, but they also serve as signaling molecules to regulate diverse biological functions. Identification of the binding proteins for the metabolites helps in the understanding of their functions beyond the classic metabolic pathways in which they are involved. We provide the protocol for synthesizing the biotin-labeled myo-inositol, which is used to identify its binding proteins by using biotin pull-down assay, given there is no available tool for the rapid screening of inositol-binding proteins in cells and in vitro systems. Biotin-labeled inositol probe therefore provides a tool to identify inositol’s sensors. For complete details on the use and execution of this protocol, please refer to. A detailed protocol for performing biotin-labeled myo-inositol pull-down assay Synthesizing and verifying biotin-conjugated myo-inositol moiety Identifying myo-inositol-binding proteins via biotin-streptavidin approach Using unlabeled myo-inositol for validating specific myo-inositol-binding proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Metabolites are not only substrates in metabolic reactions, but they also serve as signaling molecules to regulate diverse biological functions. Identification of the binding proteins for the metabolites helps in the understanding of their functions beyond the classic metabolic pathways in which they are involved. We provide the protocol for synthesizing the biotin-labeled myo-inositol, which is used to identify its binding proteins by using biotin pull-down assay, given there is no available tool for the rapid screening of inositol-binding proteins in cells and in vitro systems. Biotin-labeled inositol probe therefore provides a tool to identify inositol’s sensors.
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