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Analysis of the Phosphoinositide Composition of Subcellular Membrane Fractions

  • Deborah A. Sarkes
  • Lucia E. RamehEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)

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.

Key words

Phosphoinositide Subcellular fractionation Metabolic labeling Organelle 

Notes

Acknowledgements

This work was supported by the NIH-NIDDK grant # RO1-DK63219-06 (L.E.R.). The content does not necessarily reflect the position or the policy of the United States Government, and no official endorsement should be inferred.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.U.S. Army Research Laboratory, Sensors and Electron Devices DirectorateAdelphiUSA
  2. 2.Department of MedicineBoston University School of MedicineBostonUSA

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