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Journal of Biomolecular Screening, Vol. 9, No. 4, 343-353 (2004)
DOI: 10.1177/1087057103262841

A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

Elfrida R. Benjamin

Purdue Pharma Discovery Research, Cranbury, New Jersey

Sarah L. Haftl

Purdue Pharma Discovery Research, Cranbury, New Jersey, Glaxo SmithKline, King of Prussia, Pennsylvania

Dimitris N. Xanthos

Purdue Pharma Discovery Research, Cranbury, New Jersey, McGill University, Montreal, Canada

Gregg Crumley

Purdue Pharma Discovery Research, Cranbury, New Jersey

Mohamed Hachicha

Purdue Pharma Discovery Research, Cranbury, New Jersey

Kenneth J. Valenzano

Purdue Pharma Discovery Research, Cranbury, New Jersey

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreenTM assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

Key Words: 1,4,5-inositol-trisphosphate • G protein-coupled receptor • anion-exchange chromatography • muscarinic receptor 1 • galanin receptor 2 • metabotropic glutamate receptor 5


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