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Figure 2. Competition binding for α1D. α1D(Δ2-79) Membrane Preparation (5 and 10 μg/well) and wild-type Chem-1 Membr...
Figure 1. Saturation binding for α1D. 5 μg/well α1D(Δ2-79) Membrane Preparation was incubated with increasing amoun...
ChemiSCREEN™ Membrane Preparation Recombinant Human α1D Adrenergic Receptor with N-terminal truncation
Description:
ChemiSCREEN™ Membrane Preparation Recombinant Human α1D Adrenergic Receptor with N-terminal truncation
Trade Name:
Chemicon (Millipore)
Qty/Pk:
200 units
Product Overview:
Truncated human ADRA1D cDNA encoding α1D lacking residues 2-79
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 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. The α1D adrenergic receptor mediates smooth muscle contraction in several tissues. In the vasculature, activation of α1D increases blood pressure (Tanoue et al., 2002; Hosoda et al., 2005). In the urinary tract, α1D promotes bladder contraction. Antagonists of α1 receptors are used to treat bladder outlet obstruction, and this effect is thought to be mediated by α1D (Chen et al., 2005). The α1D adrenergic receptors has a relatively long N-terminal extracellular domain, and truncation of this domain has been shown to increase expression of the receptor at the cell surface (Pupo et al., 2003). Millipore's α1D membrane preparations, which contain a version of α1D lacking residues 2-79, 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 α1D. The membrane preparations exhibit a Kd of 0.4 nM for [3H]-prazosin. With 0.5 nM [3H]-prazosin, 5 µg/well α1D (Δ2-79) Membrane Prep typically yields greater than 5-fold signal-to-background ratio.
Application Notes:
Radioligand binding assay and GTPγS binding.
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.
Species:
Human
Quality Assurance:
Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of α1D (Δ2-79) receptor membrane prep.
SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax for [3H]-prazosin binding: 4.23 pmol/mg protein
Kd for [3H]-prazosin binding: ~0.4 nM
| 10 µg/well | 5 µg/well | |
|---|---|---|
| Signal:Background | 15.4 | 11.2 |
| Specific Binding (cpm) | 778.8 | 670.5 |
SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax for [3H]-prazosin binding: 4.23 pmol/mg protein
Kd for [3H]-prazosin binding: ~0.4 nM
Cell Type:
Chem-1
Brand Family:
Chemicon
Incubation 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 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]-prazosin. (PerkinElmer NET-823 )
Wash Buffer: 50 mM Hepes, 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 0.5 nM
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]-prazosin. (PerkinElmer NET-823 )
Wash Buffer: 50 mM Hepes, 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 0.5 nM
Host Cells:
Chem-1
Presentation:
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.
Packaging method: Membranes protein adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.
Protein Target:
alpha1D
Format:
Membranes
Ligand Type:
non-peptide
Storage Conditions:
Maintain frozen at -70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.
Target Sub-Family:
Adrenergic
Promotional Text:
For special offers Click here http://www.millipore.com/drugdiscovery/dd3/gpcrtargetsolutions
GPCR Class:
A
Product Name:
ChemiSCREEN™ Membrane Preparation Recombinant Human α1D Adrenergic Receptor with N-terminal truncation
Entrez Gene Number: