IP-One ELISA
IP1, a downstream metabolite of IP3, accumulates in cells following activation of Phospholipase C (PLC) coupled receptors. Among them, the Gq coupled GPCRs represent the most important family of receptors which can activate the β subtype of the PLC family. Other receptor types, like protein tyrosine kinase receptors, antigen or immunoglobulin receptors or collagen receptors are known to activate another PLC subtype, PLC-γ. Cisbio Bioassays developed a monoclonal antibody-based IP-One ELISA to enable simplified and highly accurate measurement of IP1.
- Cell-based functional assay
- Monoclonal antibody based
- Highly sensitive
- Simple assay protocol
- Monitor activation of PLC coupled receptors
- Validate GPCR functionality
Assay Theory:
A number of receptor families are known to induce phospholipase C (PLC) activation and trigger the Inositol phosphate (IP) cascade. Several metabolites in this pathway, including IP3, have extremely short half lives, making them difficult to accurately quantify. IP1, a downstream metabolite of IP3, accumulates in cells following Gq receptor activation and is stable in the presence of LiCl making it an ideal read out of receptor activation.
Assay Principle:
IP-One ELISA is a competitive immunoassay which uses IP1-HRP and an anti-IP1 monoclonal antibody. The kit is supplied 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.
Product Specifications:
| 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 |
Figure 1: Standard curve performed using conditions outlined in kit protocols.
Assay Performance:
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.
Figure 2: Dose response curve obtained using the CHO-M1 stable cell line stimulated with Carbachol. Cells were either cultured in 24-well or 96-well microplates during stimulation, lysed and then 50µl transferred to the pre-coated ELISA plate provided in the kit.
Ordering Info:
IP-One ELISA Reagents:
| Description | Quantity* | Cat no. | Product Insert | MSDS |
| IP-One ELISA | 96 wells | 72IP1PEA | Request MSDS | |
| IP-One ELISA | 5 X 96 wells | 72IP1PED | — | Request MSDS |
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