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.
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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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Cooke, F.T. (2010). Measurement of Phosphoinositide 3-Kinase Products in Cultured Mammalian Cells by HPLC. In: Barker, C. (eds) Inositol Phosphates and Lipids. Methods in Molecular Biology, vol 645. Humana Press. https://doi.org/10.1007/978-1-60327-175-2_12
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DOI: https://doi.org/10.1007/978-1-60327-175-2_12
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