Extraction and analysis of soluble inositol polyphosphates from yeast
Extraction and analysis of soluble inositol polyphosphates from yeast
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DOI：
10.1038/nprot.2006.337
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Saiardi, Adolfo
中科院分区：
文献类型：
--
作者：
Azevedo, Cristina;Saiardi, Adolfo
可溶性肌醇多磷酸参与许多重要细胞功能的调节。从酿酒酵母中提取和分离肌醇多磷酸的方法分为三个步骤：酵母标记、可溶性肌醇多磷酸提取和色谱分离。酵母细胞与氚化肌醇一起培养，肌醇被吸收并代谢成不同的磷酸化形式。然后用高效液相色谱法对可溶性肌醇多磷酸进行酸萃取和分馏。每个部分的放射性是通过闪烁计数来确定的。这种高灵敏度和可重复性的方法可以准确检测肌醇多磷酸谱的细微变化，并且需要不到48小时。它可以很容易地应用于其他系统，我们已经包括了两种方案的调整，一种针对哺乳动物细胞优化，另一种针对拟南芥。
Soluble inositol polyphosphates are implicated in the regulation of many important cellular functions. This protocol to extract and separate inositol polyphosphates from Saccharomyces cerevisiae is divided into three steps: labeling of yeast, extraction of soluble inositol polyphosphates and chromatographic separation. Yeast cells are incubated with tritiated inositol, which is taken up and metabolized into different phosphorylated forms. Soluble inositol polyphosphates are then acid-extracted and fractionated by high-performance liquid chromatography. The radioactivity of each fraction is determined by scintillation counting. This highly sensitive and reproducible method allows the accurate detection of subtle changes in the inositol polyphosphate profile and takes less than 48 h. It can easily be applied to other systems and we have included two adaptations of the protocol, one optimized for mammalian cells and the other for Arabidopsis thaliana.