ChemiSCREEN Membrane Preparation Recombinant Human alpha1B Adrenergic Receptor

General description

Full-length human ADRA1B cDNA encoding α_1B adrenergic

The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenoceptors (Bylund et al., 1994). The three members of the α1 subclass of adrenoceptors, α_1A, α_1B and α_1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue. Overexpression of a constitutively active α_1B mutant in the heart of transgenic mice resulted in cardiac hypertrophy with increased heart weight/body weight ratios. Analysis of α_1B knock out mice has provided evidence that α_1B is a mediator of blood pressure and aortic contractile responses induced by α₁ agonists (Milano et al., 1994). The locomotor and rewarding effects of pysochostimulants and opiates were suppressed in mice lacking α_1B-adrenergic receptors (Drouin et al. 2002). Millipore′s α_1B 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 agonists and antagonists of α_1B. The membrane preparations exhibit a Kd of 0.8 nM for [³H]-Prazosin. With 1 nM [³H]-Prazosin, 5μg/well α_1B Membrane Prep typically yields greater than 5-fold signal-to-background ratio.

Application

Radioligand binding assay and GTPγS binding.

Biochem/physiol Actions

GPCR Class: A

Protein Target: alpha1B

Target Sub-Family: Adrenergic

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of α_1B Receptor membrane prep.

	5 µg/well
Signal:Background	11.8
Specific Binding (cpm)	1171

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
B_max for [³H]-Prazosin binding: 12.9 pmol/mg protein
K_d ffor [³H]-Prazosin binding: ~0.8 nM

Specifications

Inucbation Conditions
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 Tris, pH 7.4. 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 Tris, pH 7.4, 10 mM MgCl₂, 1 mM EDTA, filtered and stored at 4°C

Radioligand: [³H]-Prazosin. (Perkin Elmer # NET823)

Wash Buffer: 50 mM Tris, 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 5-fold signal:background with ³H labeled Prazosin at 1nM

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 1 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C

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.