ChemiScreen™ Recombinant Human β2 Adrenoceptor

ChemiScreen™ Recombinant Human β2 Adrenoceptor

ChemiScreen

Human β2 adrenoceptor

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 -adrenergic receptors (Bylund et al., 1994). The β -adrenergic receptors, primarily the β2 subtype, mediate relaxation of smooth muscle in many tissues, and β 2-selective agonists are the preferred drugs for stimulating bronchodilation in the treatment of asthma and chronic obstructive pulmonary disease (Sears and Lotvall, 2005). Activation of the β-adrenergic receptors, primarily the β1 subtype and to a lesser extent the β2 subtype, acutely increases heart rate, cardiac output, and cardiac automaticity, and chronically increases cardiac myocyte apoptosis. As a result, β -adrenergic receptor antagonists (β blockers) are effective in the treatment of congestive heart failure and arrhythmia (Feldman et al., 2005). Chemicon's β2 adrenoceptor 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 β2 adrenoceptor interactions with (-)Iodocyanopindolol (ICYP). The membrane preparations exhibit a Kd of 0.51 nM for [¹²⁵I]-(-)ICYP. With 5 µg/well β2 Adrenoceptor Membrane Prep and 0.5 nM [¹²⁵I]-(-)ICYP, a greater than 40-fold signal-to-background ratio was obtained.

Radioligand binding assay, and GTPγS binding.

Human

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.

Chem-2, a suspension mammalian cell line without any endogenous β2 adrenoceptor expression.

Chem-2

• ADRBK2
• BARK2
• GRK3
• Beta-ARK-2
• EC 2.7.11.15

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 40-fold signal:background with ¹²⁵I-labeled (-)Iodocyanopindolol at 0.5 nM. Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives. Packaging method: Membranes protein were adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of β2 adrenoceptor membrane prep.

	10 µg/well	5 µg/well
Signal:Background	95	103
Specific Binding (cpm)	51393	58517

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax: 8.5 pmol/mg
Kd: 0.51 nM

Membrane Preparations

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. 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, filtered and stored at 4°C
Radioligand: [¹²⁵I]-(-) Iodocyanopindolol (Perkin Elmer # NEX189)
Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM NaCl, filtered and stored at 4°C.

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

Human