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Measurement of Phosphoinositide 3-Kinase Products in Cultured Mammalian Cells by HPLC

  • Frank T. CookeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 645)

Abstract

The phosphoinositide 3-kinase (PI3K) family catalyses the addition of a phosphate group to the D-3 position of polyphosphoinositides (PPIn). Since the discovery in the late 80s that phosphatidylinositol is phosphorylated in the D-3 position in eukaryotic cells, there has been an explosion of interest in these PPIn. Although the four D-3 PPIn (phosphatidylinositol 3-phophate (PtdIns3P), PtdIns(3,4)P 2, PtdIns(3,5)P 2, and PtdIns(3,4,5)P 3) represent only a small proportion of PPIn, production of D-3 PPIn is required for an ever-increasing number of processes. Measurement of the PPIn levels in intact cells cultured cells has been vital to our understanding of the metabolism and function of these important signalling molecules; methods are described herein that allow measurement of PPIn levels in cultured cells, with emphasis on the 3-OH PPIn.

Key words

PI3K Phosphoinositides HPLC Isotopic labelling 

Abbreviations

BSA

bovine serum albumin

cpm

counts per minute

dpm

disintegrations per minute

Etn

ethanolamine

FAF

fatty acid-free

GroP

glycerophospho

HBBSS

HEPES buffered balanced salts solution

HPLC

high performance liquid chromatography

Ins

inositol

Ptd

phosphatidyl

PI3K

phosphoinositide 3-kinase

PPIn

polyphosphoinositide

Pi

inorganic PO4

TBAS

tetrabutyl ammonium hydrogen sulphate

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Structural and Molecular BiologyUniversity College LondonLondonUK

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