Thin layer chromatography-blotting, a novel method for the detection of phosphoinositides

Phosphoinositides are believed to be involved in fundamental cellular events such as signal transduction and vesicular trafficking. Aberrant metabolisms of this lipid, caused by mutations in phosphoinositide kinases, phosphatases and lipases are known to be related to variety of human disorders such as diabetes and cancer. While the majority of such information is obtained by analyzing genetic and biochemical properties of phosphoinositide-metabolic enzymes, direct measurement of cellular content of the lipid is hindered by the lack of a simple method that is sensitive enough to measure phosphoinositides present in trace amounts in vivo. Here, we describe a novel, thin layer chromatography (TLC)-based method by which cellular phosphoinositides are separated, transferred and detected by specific phosphoinositide-binding domains. This method was applied to follow the generation of minor phosphoinositides, such as PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to insulin and to compare PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels in several cancer cell lines. The method has potential application not only in investigating the physiological roles of phosphoinositides, but also in diagnosing metabolic disease and cancer by directly assessing phosphoinositide levels in samples obtained from patients.