IP-One HTRF® assay

IP-One is the unique GPCR Gq second messenger accumulation assay.

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 receptor activation and is stable in the presence of LiCl, making it ideal for GPCR (Gq) functional assays. Cisbio Bioassays has developed and patented a highly accurate HTRF assay for measuring IP1 in 96-, 384- and 1536-well formats, enabling an HTS-compatible assay.

Features

Cell-based functional assay
Second messenger accumulation assay
Assay formats 100 µL and 20 µL
Miniaturization down to < 10µl
Compatible with fresh or frozen cells

Applications

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

Related applications and services

Cell surface receptor assays

Tag-lite ligand binding assays

HTRF® functional assays

Assay Principle
Product Specifications
Assay Performances
Part#, inserts & MSDS
Literature

The IP-One assays are competitive immunoassays that use terbium 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 just a single 1 hour incubation following cell stimulation.

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

IP-One standard curve

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

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 the receptors tested (table).

		EC50
GPCR target	Compound	HTRF®	Isotopic method
Muscarinic M1	Acetylcholine Carbachol	71 nM 296 nM	42 nM 300 nM
Vasopressin V1A	Vasopressin Vasopressin	1 nM 1.6 nM	0.4 nM 0.4 nM
Oxytocin OT (Gq)	Oxytocina	13 nM	7 nM
Histamine H2 (G16)	Amthamine	21 nM	16 nM
Purinergic P2Y1	2-methylthio ADP	6.8 nM	ND
Cholecystokinine CCK1	CCK8 Sulfate3	2 nM	43 nM
Chemokine CCRS (G16)	RANTES MIP1 α	76 nM 48 nM	26 nM ND
HupCar	Calcium	2.9 nM	ND
Endothelin Etb	Endothelin 2 Ala-endothelin3	82 nM 70 nM	83 nM 93 nM
TRHI (Gq)	TRH	0.8 nM	ND
GB1+GB2 (Gqi9)	GABA	980 nM	484 nM
mGluR 1	Quisqualate	75 nM	75 nM
mGluR5	Quisqualate	13 nM	9 nM
Muscarinic M3	Acetylcholine	20 µM	ND
Purynergic P2Y1	UTP ATP	2.1 µM 1.6 µM	ND ND

Ordering Info

Description	Cat. no	Product insert	MSDS
IP-One Tb kit - 1,000 tests	62IPAPEB	pdf	pdf
IP-One Tb kit - 20,000 tests	62IPAPEC	pdf	pdf
IP-One Tb kit -2 X 96 tests	62IPAPEF	pdf	pdf
IP-One Tb kit - 100,000 tests	62IPAPEJ	pdf	pdf
IP-One Tb kit - 1 M tests (10 packs 62IPAPEJ)	62IPAPEM	pdf	pdf

Companion products

Description	Cat. no	Product insert	MSDS
Lysis buffer 3 (IP-One kit 1,000 tests) - 13 ml	62CL3FDD	-	-
Lysis buffer 4 (IP-One kits 20, & 100,000 tests)	62CL4FDF	-	-
Lysis buffer 5 (IP-One Tb kit 1,000 tests) - 13 ml	62CL5FDD	-	-
Lysis Buffer 6 (IP-One Tb kits 20, & 100,000 tests)	62CL6FDF	-	-
IP-One calibrator	62IP1CDA	-	pdf
IP-One stimul. Buffer 5X - 8 mL	62IP1FDC	-	pdf
IP-One stimul. Buffer 5X - 100 mL	62IP1FDG	-	pdf
IP-One control	62IP1TDA	-	pdf
IP-One Tb control	62IPATDA	-	pdf
96 half-well white plate cell culture - 8 plates	66PL8ZZZ	-	-

View all
Application note8
Flyer1
Poster7
Presentation6
Scientific paper20

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IP-One : A stable second messenger of PLC-coupled receptors.

Application note8

Title

Related products

Download

HTS Instrument Discovers Low Affi nity Inhibitors of the Inositol Phosphate (IP) Signaling Pathway
An application Note from BMG LABTECH

HTRF® cAMP Dynamic 2 and IP-One Assays Using the SpectraMax M5e Multi-Detection Microplate Reader
An Application Note from Molecular Devices corporation

Functional Screening of recombinant CHO-M1 cells using the PHERAstar from BMG LABTECH
An application note from BMG LABTECH

HTRF®: IP-One Terbium-based assay performed on BMG LABTECH's PHERAstar Plus
An application Note from BMG LABTECH

cAMP and IP-One HTRF assays on the new SpectraMax M5e multi-detection
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An application Note from BMG LABTECH

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Flyer1

Title

Related products

Download

IP-One : A stable second messenger of PLC-coupled receptors.

Poster7

Title

Related products

Download

Next-Generation HTS Instrument Discovers Low Affi nity Inhibitors of the Inositol Phosphate (IP) Signaling Pathway.
Dell EJ, Tardieu JL, Degorce F, Fejtl M
SLAS 2012, San Diego, California, USA

Presentation6

Title

Related products

Download

Two GPCR Case Studies with the IP-One Eu and Tb Assay Kits. A Positive Modulator and an Inverse Agonist Project
Martin Graf, F
SBS 14th annual conference. April 2008, St Louis, USA

qHTS for Identifying the Cell Membrane Permeable Inositol Monophosphatase Inhibitors with HTRF® IP-One Reagents
Zheng W
HTRF® 3rd Annual Symposium 2007, Sonoma, CA, USA

Case Study: HTS of GPR-54 with IP-One Assay
Glicksman MA
Assays and Cellular Targets 2006

IP-One & cAMP HTRF® Functional Cell-based Screening
Bader B, Klotz M, Zopf D, Glienke J, Martinez S
IIR Conference 2006, London, United kingdom

Scientific paper20

Title

Related products

Download

Biased agonism of the calcium-sensing receptor
Thomsen AR, Hvidtfeldt M, Bräuner-Osborne H.
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Trans-activation between 7TM domains: implication in heterodimeric GABAB receptor activation.
Monnier C, Tu H, Bourrier E, Vol C, Lamarque L, Trinquet E, Pin JP, Rondard P.
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Monitoring Gq-coupled receptor response through inositol phosphate quantification with the IP-One assay.
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Zhang JY, Kowal DM, Nawoschik SP, Dunlop J, Pausch MH, Peri R.
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Cassutt KJ, Orsini MJ, Abousleiman M, Colone D, Tang W
J Biomol Screen. 2007;12(2):285-7.