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Competition binding for ETB. ETB Membrane Preparation (10 μg/well) or Wild-Type Chem-1 membrane preparation (WT; Millipore Catalog # HTS000MC1) was ...
Saturation binding for ETB. 10 μg/well ETB Membrane Preparation was incubated with increasing amount of [125I]Endothelin-1 in the absence ...
ChemiSCREEN™ Membrane Preparation Recombinant Human ETB Endothelin Receptor
Description:
ChemiSCREEN™ Membrane Preparation Recombinant Human ETB Endothelin Receptor
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Qty/Pk:
200 units
Product Overview:
Human ETB isoform 1
Background Information:
The endothelin family of peptides are potent vasoconstrictors synthesized by endothelial cells in both constitutive and inducible pathways. The biological actions of endothelins are mediated by two GPCRs, ETA and ETB (Davenport, 2002). Whereas ETA is the predominant receptor on smooth muscle cells and thus is the primary mediator of the vasoconstrictor activity, ETB is found on endothelial cells and mediates vasodilation (Nilsson et al., 1997). In addition, ETB has also been linked to renal failure, congestive heart failure, atherosclerosis, and pulmonary hypertension (D’Orléans-Juste, et al., 2002). A mutation in ETB has been found to cause Hirschsprung disease-2, a degenerative disease of the digestive tract (Puffenberger et al, 1994). Millipore’s cloned human ETB expressing cell line is made in the CHO host, which supports high levels of recombinant ETB expression on the cell surface. Thus, the ETB Membrane Preparation is an ideal tool for screening for compounds binding to ETB. The membrane preparations exhibit a Kd of 0.03-0.06 nM for [125I]-endothelin-1. With 0.2 nM [125I]-endothelin-1, 10 μg/well ETB Membrane Prep 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 ETB membrane prep.
SPECIFICATIONS: 1 unit = 10 μg membrane preparation
Bmax: 0.49 pmol/mg
Kd: 0.03-0.06 nM
| 10 µg/well | |
|---|---|
| Signal:Background | 7.4 |
| Specific Binding (cpm) | 5704 |
SPECIFICATIONS: 1 unit = 10 μg membrane preparation
Bmax: 0.49 pmol/mg
Kd: 0.03-0.06 nM
Cell Type:
CHO
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: [125I]Endothelin-1 (Perkin Elmer # NEX259)
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 an unit is the amount of membrane that will yield greater than 5-fold signal:background with 125I-labeled Endothelin-1 at 0.25 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: [125I]Endothelin-1 (Perkin Elmer # NEX259)
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 an unit is the amount of membrane that will yield greater than 5-fold signal:background with 125I-labeled Endothelin-1 at 0.25 nM.
Host Cells:
CHO (Chinese hamster ovary cells)
Protein Target:
ETB
Presentation:
One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 5-fold signal:background with 125I-labeled Endothelin-1 at 0.25 nM.
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.
Ligand Type:
peptide
Storage Conditions:
Store 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:
Endothelin
Promotional Text:
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GPCR Class:
A
Protein or Enzyme Type:
Membrane Preparations
Product Name:
ChemiSCREEN™ Membrane Preparation Recombinant Human ETB Endothelin Receptor
Entrez Gene Number: