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HTRF IP-One Assay Performed on the PHERAstar and RUBYstar Plate Readers

ion is irreversible. This of course has tended to make its detection highly inaccurate. IP3 induces a transient calcium release in the cell. Calcium sensing, through a very remote indicator, can allow the high throughput investigation of GPCR activity. To date, there has been no widespread use of IP assays in HTS, given its extremely challenging implementation. For lack of better options, the reference method consists of an assay which detects the accumulation of the cascades different inositol phosphates (IP3, IP4, IP2 and IP1) after the radioactive precursors uptake and a separation by affinity.

Recently Cisbio launched a major new kit, HTRF IP-One, for Gq pathway investigation under HTS conditions. The IP-One kit allows the quantification of the cellular accumulation of inositol 1 phosphate (IP1).

This application note focuses on the IP1 assay which has been validated on a broad and representative selection of Gq coupled receptors using the dedicated HTRF plate reader RUBYstar and the multimode plate reader PHERAstar from BMG LABTECH. These HTRF certified compatible readers are capable to simultaneously detect the fluorescence at two wavelengths (620 nm and 665 nm) and further signal ratioing also enables the technology to overcome interference from the medium or from compounds. Such technological efficiency, combined with the use of a high-affinity monoclonal antibody, give the assay all the qualities necessary for an HTS tool, particularly in terms of sensitivity, robustness and reliability.


Assay Principle
As previously mentioned, IP3, though, being the top choice as a secondary messenger for the Gq signaling pathway, ne
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