ChemiSCREEN Human Angiotensin II Receptor, Type I Membrane Preparation

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

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: AT1

Target Sub-Family: Angiotensin

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

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

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.

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

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