title: Analysis of the Phosphoinositide Composition of Subcellular Membrane Fractions.
abstract: Phosphoinositides play critical roles in the transduction of extracellular signals through the plasma membrane and also in endomembrane events important for vesicle trafficking and organelle function (Di Paolo and De Camilli, Nature 443(7112):651-657, 2006). The response triggered by these lipids is heavily dependent on the microenvironment in which they are found. HPLC analysis of labeled phosphoinositides allows quantification of the levels of each phosphoinositide species relative to their precursor, phosphatidylinositol. When combined with subcellular fractionation techniques, this strategy allows measurement of the relative phosphoinositide composition of each membrane fraction or organelle and determination of the microenvironment in which each species is enriched. Here, we describe the steps to separate and quantify total or localized phosphoinositides from cultured cells.
authors.affiliation: U.S. Army Research Laboratory, Sensors and Electron Devices Directorate, Adelphi, MD, 20783, USA.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Sarkes
authors.affiliation: Department of Medicine, Boston University School of Medicine, 650 Albany Street, Rm 830, Boston, MA, 02118, USA. rameh@bu.edu.
authors.forename: Lucia E
authors.initials: LE
authors.lastname: Rameh
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D010716:Phosphatidylinositols / Q000737:chemistry; D013194:Staining and Labeling; D013347:Subcellular Fractions; D014316:Tritium / Q000737:chemistry
chemical_list: D010716:Phosphatidylinositols; D014316:Tritium
keywords: Metabolic label ing; Organelle; Phosphoinositide; Subcellular fractionation