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Figure 1. Saturation binding for α2B. 5 µg/well α2B Membrane Preparation was incubated with increasing amount of 3H-labeled Ra ...
Figure 2. Competition binding for α2B. 5 µg/well α2B Membrane Preparation and wild-type Chem-1 Membrane Preparation (Chemicon c ...
ChemiSCREEN™ Human α2B Adrenergic Receptor Membrane Preparation
Description:
ChemiSCREEN™ Human α2B Adrenergic Receptor Membrane Preparation
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Product Overview:
Full-length human ADRA2B transcript cDNA encoding α2B.
Background Information:
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 α2A, α2B, and α2C, couple primarily to Gi to inhibit cAMP production, and play an important role in regulation of cardiovascular and CNS function. Experiments with α2A-selective agonists and mice lacking α2A demonstrate that activation of α2A results in hypotension, sedation, analgesia, and hypothermia (Kable et al., 2000). Chemicon's α2B 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 α2B. The membrane preparations exhibit a Kd of 11.5 nM for [3H]-Rauwolscine. With 15 nM [3H]-Rauwolscine, 5 µg/well α2B Membrane Prep typically yields greater than 30-fold signal-to-background ratio.
Species Reactivity:
Human
Usage Statement:
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.
Cell Membrane Type:
GPCR
Alternate Names:
alpha2B
Descriptive Text:
Host Cells:
Chem-1, an adherent mammalian cell line with minimum amount of endogenous α2B.
Cell Type:
Chem-1
Gene Symbol:
- ADRA2B
- ADRA2L1
- ALPHA2BAR
- ADRA2RL1
- ADRARL1
Presentation:
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 Rauwolscine at 15 nM
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Quality Assurance:
Table 1. Signal:background and specific binding values obtained in a competition binding assay with α2B membrane prep and unlabeled rauwolscine.
SPECIFICATIONS: 1 unit = 5 µg
Bmax for [3H]-rauwolscine binding: 154 pmol/mg protein
Kd for [3H]-rauwolscine binding: ~11.5 nM
| 5 µg/well | |
|---|---|
| Signal:Background | 64.7 |
| Specific Binding (cpm) | 14646 |
SPECIFICATIONS: 1 unit = 5 µg
Bmax for [3H]-rauwolscine binding: 154 pmol/mg protein
Kd for [3H]-rauwolscine binding: ~11.5 nM
Protein or Enzyme Type:
GPCR Membrane Preparations
Incubation 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: [3H]-Rauwolscine (Perkin Elmer#: NET-722)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.
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]-Rauwolscine (Perkin Elmer#: NET-722)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.
Species:
Human
Storage Conditions:
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.