A miniaturized column chromatography method for measuring receptor-mediated inositol phosphate accumulation
| Autor: | Kenneth J. Valenzano, Mohamed Hachicha, Elfrida R. Benjamin, Dimitris N. Xanthos, Sarah L. Haftl, Gregg Crumley |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
0301 basic medicine
DNA Complementary Inositol Phosphates Receptor Metabotropic Glutamate 5 CHO Cells Inositol 1 4 5-Trisphosphate Phospholipase Receptors Metabotropic Glutamate 01 natural sciences Biochemistry High-performance liquid chromatography Analytical Chemistry Cell Line Receptors G-Protein-Coupled 03 medical and health sciences chemistry.chemical_compound Column chromatography Cricetinae Animals Humans Centrifugation Inositol Inositol phosphate chemistry.chemical_classification Chromatography Miniaturization Base Sequence Receptor Muscarinic M1 Inositol trisphosphate receptor Chromatography Ion Exchange Recombinant Proteins 0104 chemical sciences Rats Receptor Galanin Type 2 010404 medicinal & biomolecular chemistry 030104 developmental biology chemistry Astrocytes Molecular Medicine Intracellular Biotechnology Signal Transduction |
| Zdroj: | Journal of biomolecular screening. 9(4) |
| ISSN: | 1087-0571 |
| Popis: | Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP(3)), 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 AlphaScreen assays, offer higher throughput. However, these techniques rely on measurement of IP(3) itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP(3). 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 IP(3) 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. |
| Databáze: | OpenAIRE |
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