A multi-well filtration assay for quantitation of inositol phosphates in biological samples.
The quantitation of inositol phosphates (IPs), mediators of certain signal transduction processes, typically involves laborious and time consuming conventional ion-exchange chromatography procedures. We have developed a high throughput microtiter plate-based IP assay that utilizes vacuum rather than gravitational flow and has significant advantages over existing methods. The response of recombinant HEK-293 cells expressing human LHRH receptor cDNA to LHRH agonists was used as a model system to develop the assay conditions. Cell lysates containing labeled IPs were applied in 96-well plates fitted with filtration discs containing regenerated Dowex AGI-X8 resin. Specifically bound inositol phosphates were eluted with 1 M ammonium formate in 0.1 M formic acid directly into a fresh 96-well plate and an aliquot of the eluate from each well is transferred into a 96-well plate and counted. The results were comparable to those obtained with the conventional column method and the variation among replicates was significantly improved. This assay facilitates rapid quantitation of inositol phosphates from a large number of samples with relative ease and reduced generation of radioactive waste.
Chengalvala
M
M
Women's Health Research Institute, Wyeth-Ayerst Research, Radnor, PA 19087, USA. chengam@war.wyeth.com
Kostek
B
B
Frail
D E
DE
D002478:Cells, Cultured; D002845:Chromatography; D004305:Dose-Response Relationship, Drug; D005374:Filtration / Q000295:instrumentation* / Q000379:methods*; D005561:Formates / Q000737:chemistry; D006801:Humans; D007295:Inositol Phosphates / Q000032:analysis*
D005561:Formates; D007295:Inositol Phosphates; C030544:formic acid