Scintillation proximity assay of inositol phosphates in cell extracts: High-throughput measurement of G-protein-coupled receptor activation
Section snippets
Detection of radioactive Ins and InsP standards
d-Myo-[2-]inositol 1-phosphate ([]Ins-1-P; 5–20 Ci/mmol), d-myo-[2-]inositol 1,4-bisphosphate ([]Ins-1,4-P2; 2–10 Ci/mmol), and d-myo-[1-]inositol 1,4,5-trisphosphate ([]Ins-1,4,5-P3; 15–30 Ci/mmol) were from NEN and were supplied in aqueous 10 mM ammonium phosphate, pH 8.0. Myo-[1,2-]inositol ([]Ins; NEN) was supplied in water with a specific activity of 70–80 Ci/mmol. Yttrium silicate (YSi) scintillation proximity assay (SPA) beads were supplied by Amersham Biosciences as a 100
Detection of []myo-inositol 1-phosphate versus []myo-inositol using YSi SPA beads
Yttrium silicate glass beads, impregnated with cerium, are marketed by Amersham Biosciences for use in a SPA for RNA. These SPA beads carry a net positive charge and so, in principle, should bind a negatively charged InsP more efficiently than neutral Ins. Indeed, we found that with YSi SPA beads, detection of []Ins-1-P was approximately 20-fold more efficient than for []Ins (Table 1). Addition of unlabeled Ins-1-P reduced the number of counts recorded from YSi SPA beads mixed with [
Acknowledgements
We acknowledge Dr. John York for valuable discussion of this work, Dr. Qingyun Liu for provision of the NPFFR2-expressing cell line, Dr. Pierre Mallorga for discussions concerning muscarinic receptor antagonists, and Drs. Lawrence Kuo and Pierre Mallorga for critical evaluation of the manuscript.
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