A femtomole-sensitivity mass assay for inositol hexakisphosphate.

Andrew J. Letcher, Michael J. Schell, Robin F. Irvine

Research output: Contribution to journalArticle

Abstract

Inositol hexakisphosphate (InsP(6)) is an important component of cells, and its mass levels are usually assayed by either (a) equilibrium labelling of cell cultures with radiolabelled inositol or (b) by a variety of mass assays of differing sensitivities and ambiguities. Here, we describe a mass assay for InsP(6) that is based on phosphorylating InsP(6) with [(32)P]-ATP to 5-(PP)InsP(5) using a recombinant Giardia InsP(6) kinase and quantification of the radiolabelled 5-[(32)P](PP)InsP(5) product by anion exchange HPLC with an internal [(3)H]-(PP)InsP(5) standard. Interference with the enzyme reaction by other factors in the tissue extract is corrected for by assay of identical aliquots of tissue spiked with known amounts of InsP(6). This assay only measures InsP(6) (and not other inositol phosphates), and although it is simple in principle and requires no dedicated or specialised equipment, it is quite time-consuming. But the assay is unambiguous and is capable of quantifying accurately as little as 10 fmol of InsP(6) in a cell extract.

Original languageEnglish (US)
Pages (from-to)61-71
Number of pages11
JournalMethods in molecular biology (Clifton, N.J.)
Volume645
DOIs
StatePublished - 2010
Externally publishedYes

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Giardia
Phytic Acid
Inositol Phosphates
Tissue Extracts
Inositol
Cellular Structures
Cell Extracts
Anions
Phosphotransferases
Cell Culture Techniques
Adenosine Triphosphate
High Pressure Liquid Chromatography
Equipment and Supplies
Enzymes

ASJC Scopus subject areas

  • Medicine(all)

Cite this

A femtomole-sensitivity mass assay for inositol hexakisphosphate. / Letcher, Andrew J.; Schell, Michael J.; Irvine, Robin F.

In: Methods in molecular biology (Clifton, N.J.), Vol. 645, 2010, p. 61-71.

Research output: Contribution to journalArticle

Letcher, Andrew J. ; Schell, Michael J. ; Irvine, Robin F. / A femtomole-sensitivity mass assay for inositol hexakisphosphate. In: Methods in molecular biology (Clifton, N.J.). 2010 ; Vol. 645. pp. 61-71.
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