GPCR Gq stimulation is known to induce phospholipase C (PLC) activation and trigger the Inositol phosphate (IP) cascade.
IP1, a downstream metabolite of IP3, accumulates in cells following Gq recepetor activation and is stable in the presence of LiCl, making it an ideal GPCR (Gq) functional assay.
Cisbio Bioassays has developed a monoclonal antibody-based IP-One ELISA to enable simplified and highly accurate measurement of IP1.
IP-One ELISA is a competitive immunoassay which uses IP1-HRP and an anti-IP1 monoclonal antibody. The kit comes with 96-well microplates pre-coated with an anti-mouse antibody. Cells are stimulated in the presence of LiCl, causing the accumulation of IP1 upon receptor activation. The protocol consists of two steps following cell stimulation: addition of ELISA components and addition of TMB, the HRP substrate. The HRP reaction is stopped and the optical density (OD) read at 450nm.
IP1, a downstream metabolite of IP3, accumulates in cells following Gq receptor activation. It is stable in the presence of LiCl, making it ideal for GPCR (Gq) functional assay.
| Detection limit | 10 nM |
|---|---|
| Dynamic range | 10-3000 nM |
| EC50 | 110 nM |
| Specificity | No cross-reactivity with 50 µM Myo-inositol, PIP2, IP2, IP3, IP4 or PIP3 |
The Cisbio Bioassays IP-One ELISA is compatible with cell lysates, enabling live cell functional assays. Cells can be stimulated using a variety of plate formats prior to the IP-One ELISA detection steps. Figure 2 shows the results of an IP-One ELISA performed on cells stimulated in either a 24- or 96-well format. Cells were lysed and then transferred to the ELISA plate provided in the kit. We used 80,000 cells per well in the 96-well plates and 400,000 cells per well in the 24-well format. Both cell stimulation formats produced similar results, with low standard deviations.
Dose response curve obtained using the CHO-M1 stable cell line stimulated with Carbachol. Cells were cultured in either 24-well or 96-well microplates during stimulation, lysed, and then 50µl transferred to the kit's pre-coated ELISA plate.
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