IP-One HTRF^® Assay

Gq coupled receptor stimulation is 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 receptor activation and is stable in the presence of LiCl.

Cisbio Bioassays developed and patented a highly accurate HTRF assay for measuring IP1 in 96-, 384- and 1536-well formats providing for a Gq coupled receptor functional assay.

Features:

Proven, cell-based functional assay
Simple and rapid assay protocol
Stable EC50 from 1 hour to 7 days
Miniaturization down to
< 10µl
Large screening window with Z' greater that 0.85

Applications:

Gq coupled receptor activation
Assess agonists, antagonists, allosteric modulators and inverse agonists
Detection of slow acting agonist in functional assay

Assay Principle
Product Specifications
Assay Performance
Ordering Info

Assay Principle:

The IP-One assays are competitive immunoassays that use cryptate-labeled anti-IP1 MAb and d2-labeled IP1. LiCl is added to the cell stimulation buffer causing the accumulation of IP1 upon receptor activation. The assay can be run in a single microplate and requires only a single 1 hour incubation following cell stimulation.

Product Specifications:

Detection Limit:	5 nM
Dynamic Range:	5 - 3000 nM
S/B:	20 (depending on reader performance)
EC50:	100 nM
Z':	0.91 (20 µL, 384 wells)
Specificity:	No cross-reactivity with 50 µM Myo-inositol, PIP2, IP2, IP3, IP4 or PIP3
Sample types:	Living or frozen cells
Additive tolerance:	> 5% DMSO

IP1 standard curve performed using protocol outlined in IP-One Tb assay product insert.

Assay Performance:

The HTRF IP-One kit is a proven assay for assessing Gq coupled receptor activation. A side-by-side comparison was run between the HTRF IP-One kit and an isotopic inositol cascade method using a range of Gq coupled receptors and their associated agonist. The EC50 values calculated using HTRF and isotopic methods were comparable for all receptors that were tested (Table 1).

			EC₅₀
GPCR target (G protein)	Cell line	Agonist	HTRF	Isotopic method
Muscarinic M1(Gq)	CHO-K1	Acetylcholine^a Carbachol^a	71 nM 296 nM	42 nM 300 nM
Vasopressin V1A (Gq)	CHO-K1	Vasopressin^a Vasopressin^b	1 nM 1.6 nM	0.4 nM 0.4 nM
Oxytocin OT (Gq)	CHO-K1	Oxytocin^a	13 nM	7 nM
Histamine H2 (G16)	CHO-K1	Amthamine^a	21 nM	16 nM
Purinergic P2Y1 (Gq)	1321N1	2-methylthio ADP^a	6.8 nM	ND
Cholecystokinine CCK1 (Gq)	1321N1	CCK8 Sulfate³	2 nM	43 nM
Chemokine CCRS (G16)	CHO-K1	RANTES^a MIP1 α^a	76 nM 48 nM	26 nM ND
HupCar (Gq)	CCL39	Calcium^a	2.9 nM	ND
Endothelin Etb (Gq)	CHO-K1	Endothelin 2^a Ala-endothelin³	82 nM 70 nM	83 nM 93 nM
TRHI (Gq)	CHO-K1	TRH^a	0.8 nM	ND
GB1+GB2 (Gqi9)	HEK293	GABA^a	980 nM	484 nM
mGluR 1 (Gq)	HEK293	Quisqualate^a	75 nM	75 nM
mGluR5 (Gq)	HEK293	Quisqualate^a	13 nM	9 nM
Muscarinic M3 (Gq)	HEK293	Acetylcholine^a	20 µM	ND
Purynergic P2Y1 (Gq)	HEK293	UTP^a ATP^a	2.1 µM 1.6 µM	ND ND
(a) Tested with adherent cells. (b) Tested with suspended cells.

Ordering Info:

IP-One Assay Reagents:

Description	Quantity	Cat no.	Product Insert	MSDS
IP-One Tb kit - 384-well assay format (20 µl)	1,000 tests	62IPAPEB	pdf	pdf
	20,000 tests	62IPAPEC		pdf
	100,000 tests	62IPAPEJ		pdf
	1,000,000 tests	62IPAPEM		pdf

Description	Quantity	Cat no.	Product Insert	MSDS
IP-One Tb kit - 96-well assay format (100 µl)	2x96 tests	62IPAPEF	pdf	-

Companion Products:

Description	Cat no.	MSDS
IP-One calibrator	62IP1CDA	pdf
IP-One control	62IP1TDA	-
IP-One stimulation buffer 5X (8 ml)	62IP1FDC	pdf
IP-One stimulation buffer 5X (100 ml)	62IP1FDG	-
Lysis buffer (13 ml)	62CL5FDD	-
Lysis buffer (200 ml)	62CL6FDF	-