Inositol Triphosphate Assays

Whereas most HTS labs utilize gene reporter or calcium fluorophore technologies to detect Gq- and/or Gi-coupled receptors, some technologies detect inositol-triphosphate (IP3) formation in cells. Historically, IP3 assays depended on [3H]inositol loading into cells. Radioactivity was then detected in aqueous and organic fractions after column separation. This technology is not readily amenable for HTS. IP3 formation is determined by measuring the displacement of a labeled IP3 analog from an IP3 binding protein.

Kits are available from Amersham, PerkinElmer (AlphaScreen) and DiscoveRx (enzyme complementation). These assays may prove useful in secondary screening. One disadvantage of IP3 detection is the limited timeframe during which its production can be detected due to the kinetics of IP3 formation after receptor stimulation which is extremely fast. Therefore, care must be taken to ensure proper timing as well as fast and complete lysis of stimulated cells.

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