Fedeli Valeria, Saiardi Adolfo
Laboratory for Molecular Cell Biology, University College London, London, UK.
Methods Mol Biol. 2025;2972:29-38. doi: 10.1007/978-1-0716-4799-8_3.
Polyacrylamide gel electrophoresis (PAGE) is a versatile technique widely used in molecular biology for the separation of biomolecules based on size and charge. While it is traditionally applied to proteins and nucleic acids, recently, PAGE has been adapted for the analysis of inositol polyphosphates and their pyrophosphate derivatives, which play multiple roles in cellular signaling. Studying these molecules presents analytical challenges due to their small size, high charge density, and low concentrations in biological samples. In this work, we describe an optimized PAGE protocol for the separation and detection of inositol polyphosphates and inositol pyrophosphate. The method uses high-concentration polyacrylamide gels and toluidine blue staining to achieve sensitive detection at nanomolar levels. The simplicity and cost-efficiency of the approach make it accessible to most laboratories. This method provides a reliable tool for investigating inositol-phosphate-based signaling pathways and their involvement in cellular processes.
聚丙烯酰胺凝胶电泳（PAGE）是一种用途广泛的技术，在分子生物学中被广泛用于根据大小和电荷分离生物分子。虽然它传统上应用于蛋白质和核酸，但最近，PAGE已被用于分析肌醇多磷酸及其焦磷酸衍生物，这些物质在细胞信号传导中发挥多种作用。由于它们尺寸小、电荷密度高且在生物样品中的浓度低，研究这些分子面临分析挑战。在这项工作中，我们描述了一种优化的PAGE方案，用于分离和检测肌醇多磷酸和肌醇焦磷酸。该方法使用高浓度聚丙烯酰胺凝胶和甲苯胺蓝染色，以实现纳摩尔水平的灵敏检测。该方法的简单性和成本效益使大多数实验室都能采用。此方法为研究基于肌醇磷酸的信号通路及其在细胞过程中的作用提供了可靠工具。
