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IP-One HTRF® assay kit

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

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.

GPCR IP1 assay protocol

IP one graphs
IP one graphs
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 ATP 1.6 µM ND

 

Ordering Info

DescriptionCat. noProduct insertMSDS
IP-One Tb kit - 1,000 tests62IPAPEB
IP-One Tb kit - 20,000 tests62IPAPEC
IP-One Tb kit -2 X 96 tests62IPAPEF
IP-One Tb kit - 100,000 tests62IPAPEJ
IP-One Tb kit - 1 M tests (10 packs 62IPAPEJ)62IPAPEM
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Companion products

DescriptionCat. noProduct insertMSDS
Lysis buffer 5 (IP-One Tb kit 1,000 tests) - 13 ml62CL5FDD
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Lysis Buffer 6 (IP-One Tb kits 20, & 100,000 tests)62CL6FDF
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IP-One calibrator62IP1CDA
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IP-One stimul. Buffer 5X - 8 mL62IP1FDC
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IP-One stimul. Buffer 5X - 100 mL62IP1FDG
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IP-One Tb control62IPATDA
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96 half-well white plate cell culture - 8 plates66PL8ZZZ
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