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A Femtomole-Sensitivity Mass Assay for Inositol Hexakisphosphate

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 645)

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 [32P]-ATP to 5-(PP)InsP 5 using a recombinant Giardia InsP 6 kinase and quantification of the radiolabelled 5-[32P](PP)InsP 5 product by anion exchange HPLC with an internal [3H]-(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.

Key words

Inositol Inositol phosphate Inositol hexakisphosphate Phytic acid Phytate Mass assay 
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Notes

Acknowledgments

This work was supported by the Wellcome Trust and the Royal Society. We thank Kevin Brindle for helpful discussions.

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© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of PharmacologyUniversity of CambridgeCambridgeUK
  2. 2.Department of PharmacologyUniformed Services University of the Health SciencesBethesdaUSA

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