Description
Utilizing a competitive immunoassay format, the HTRF IP-One assays detect the accumulation of inositol monophosphate (IP1), a stable downstream metabolite of IP3 induced by activation of a phospholipase C (PLC) cascade. This kit allows direct characterization of all types of compounds acting on Gq-coupled receptors in either adherent or suspension cells.
From high-throughput screening (HTS) to structure-activity relationships (SAR), IP-One has been shown to offer superior benefits over more traditional assays.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
The kit is based on a competitive format involving a specific antibody labeled with cryptate (donor) and IP1 coupled to d2 (acceptor). Native IP1 produced by cells competes with d2-labeled IP1 for binding to monoclonal anti-IP1 Tb2+ cryptate. LiCl is added to the cell stimulation buffer, causing IP1 to accumulate upon receptor activation.
Assay protocol
The IP-One assay features a streamlined protocol with just two incubation steps: Cell stimulation by the ligand or target compounds IP1 detection using HTRF reagentsThis protocol requires only a single, one-hour incubation period following cell stimulation.
Specificity of IP-One Gq assay
IP-One ensures signal specificity, measuring only IP-1, an analyte that is solely produced by the action of the phospholipase enzyme (PLC) and mediated by GPCRs.
In contrast, calcium responses are complex in nature. Calcium release is frequently triggered by non-G protein mechanisms, including Ca2+ permeable channels, calcium pumps, calcium transporters.
Standard curve characteristics
Assay Window, sensitivity, reproducibility and specificity : IP-One makes no concession !
