Alpha2A Membrane Preparation

Application

Radioligand binding assay and GTPγS binding.

Human alpha2A GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Biochem/physiol Actions

Target Sub-Family: Adrenergic

GPCR Class: A

Protein Target: alpha2A

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.

General description

Full-length human ADRA2A transcript variant 1 cDNA encoding α_2A

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 α₂ adrenergic receptor subfamily members, consisting of α_2A, α_2B, and α_2C, couple primarily to Gi to inhibit cAMP production, and play an important role in regulation of cardiovascular and CNS function. Experiments with α_2A-selective agonists and mice lacking α_2A demonstrate that activation of α_2A results in hypotension, sedation, analgesia, and hypothermia (Kable et al., 2000). Chemicon′s α_2A 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 at α_2A. The membrane preparations exhibit a Kd of 5.4 nM for [³H]-MK-912. With 5 nM [³H]-MK-912, 5 µg/well α_2A Membrane Prep typically yields greater than 20-fold signal-to-background ratio.

Legal Information

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

Physical form

One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 20-fold signal:background with 3H-labeled MK-912 at 5 nM

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with α_2A membrane prep and unlabeled rauwolscine.

Specifications

Inucbation ConditionsMembranes 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: [3H] MK-912 (Perkin Elmer#: NET-1059)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.

Storage and Stability

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