HTS158M
ChemiSCREEN Membrane Preparation Recombinant Human α1B Adrenergic Receptor
Human alpha1B GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
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About This Item
UNSPSC Code:
41106514
eCl@ss:
32161000
NACRES:
NA.41
Recommended Products
biological source
human
Quality Level
recombinant
expressed in Chem-1 cells
manufacturer/tradename
ChemiScreen
Chemicon®
technique(s)
ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable
NCBI accession no.
UniProt accession no.
shipped in
dry ice
General description
Full-length human ADRA1B cDNA encoding α1B 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, α1A, α1B and α1D, 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 α1B mutant in the heart of transgenic mice resulted in cardiac hypertrophy with increased heart weight/body weight ratios. Analysis of α1B knock out mice has provided evidence that α1B 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 α1B-adrenergic receptors (Drouin et al. 2002). Millipore′s α1B 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 α1B. The membrane preparations exhibit a Kd of 0.8 nM for [3H]-Prazosin. With 1 nM [3H]-Prazosin, 5μg/well α1B 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 α1B Receptor membrane prep.
SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax for [3H]-Prazosin binding: 12.9 pmol/mg protein
Kd ffor [3H]-Prazosin binding: ~0.8 nM
| 5 µg/well | |
|---|---|
| Signal:Background | 11.8 |
| Specific Binding (cpm) | 1171 |
SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax for [3H]-Prazosin binding: 12.9 pmol/mg protein
Kd 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
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
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.
Storage Class Code
12 - Non Combustible Liquids
WGK
WGK 2
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Regulatory Listings
Regulatory Listings are mainly provided for chemical products. Only limited information can be provided here for non-chemical products. No entry means none of the components are listed. It is the user’s obligation to ensure the safe and legal use of the product.
JAN Code
HTS158M:
Certificates of Analysis (COA)
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Find documentation for the products that you have recently purchased in the Document Library.
Myocardial expression of a constitutively active alpha 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy.
Milano, C A, et al.
Proceedings of the National Academy of Sciences of the USA, 91, 10109-10113 (1994)
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