ChemiSCREEN Human Angiotensin II Receptor, Type I Membrane Preparation

General description

Angiotensin II (Ang II), an octapeptide produced by cleavage of angiotensinogen by angiotensin-converting enzyme, plays a fundamental role in cardiovascular homeostasis. Two GPCRs, AT₁ and AT₂, mediate the effects of AngII, although AT₁ is primarily responsible for the effects of Ang II on renal function and development, thirst, electrolyte and water balance, and arterial blood pressure. Binding of Ang II to AT₁ activates both G_q and G_i (De Gasparo et al., 2000). Chemicon′s AT₁ membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of antagonists of AT₁ interactions and its ligands. The membrane preparations exhibit a Kd of 0.36 nM for [¹²⁵I]-Sar1-Ile8-Angiotensin II. With 0.3 nM [¹²⁵I]-Sar1-Ile8-Angiotensin II, 5 µg/well AT₁ Membrane Prep yields greater than 50-fold signal-to-background ratio.

Full-length human cDNA encoding AT₁

Application

Radioligand binding assay and GTPγS binding.

Biochem/physiol Actions

GPCR Class: A

Protein Target: AT1

Target Sub-Family: Angiotensin

Quality

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of ATaub1 membrane prep:
tabletrth align="left"/tdth align="center"10 µg/well/tdth align="center"5 µg/well /td/trtrtd align="left" Signal:Background/tdtd align="center" 115.5/tdtd align="center" 92.8/td/trtrtd align="left" Specific Binding (cpm) /tdtd align="center"46,458/tdtd align="center"43,007 /td/tr/table

SPECIFICATIONS: 1 unit = 5 µg
Bmax for [¹²⁵I] Angiotensin II binding: 5.53 pmol/mg protein;
Kd for [¹²⁵I] Angiotensin II binding: ~ 0.36 nM

Specifications

Inucbation Conditions
Membranes are mixed with radioactive ligand and unlabeled competitor (see Figures 1 and 2 for concentrations tested) in binding buffer in a nonbinding 96-well plate, and incubated for 1-2 h. Prior to filtration, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. Binding reaction is transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate is dried and counted.
Binding buffer: 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 0.2% BSA, filtered and stored at 4°C
Radioligand: [¹²⁵I]-Sar1-Ile8-Angiotensin II. (Perkin Elmer#:NEX-248 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.
One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 50-fold signal:background with ¹²⁵I labeled Sar1-Ile8-Angiotensin II at 0.3 nM

Physical form

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80oC.

Storage and Stability

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.