ChemiSCREEN Membrane Preparation Recombinant Human AT₂ Angiotensin II Receptor

FUNCTION: SwissProt: P50052 # Receptor for angiotensin II. Cooperates with MTUS1 to inhibit ERK2 activation and cell proliferation.
SIZE: 363 amino acids; 41184 Da
SUBUNIT: Interacts with MTUS1.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: In adult, highly expressed in myometrium with lower levels in adrenal gland and fallopian tube. Very highly expressed in fetal kidney and intestine.
SIMILARITY: SwissProt: P50052 ## Belongs to the G-protein coupled receptor 1 family.

Radioligand binding assay

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

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

RECOMMENDED ASSAY 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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) 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]-CGP42112. (Perkin Elmer # NEX-324 )

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 CGP42112 at 0.25 nM

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