ChemiSCREEN Membrane Preparation Recombinant Human ET_B Endothelin Receptor

Human ET_B isoform 1

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, ET_A and ET_B (Davenport, 2002). Whereas ET_A is the predominant receptor on smooth muscle cells and thus is the primary mediator of the vasoconstrictor activity, ET_B is found on endothelial cells and mediates vasodilation (Nilsson et al., 1997). In addition, ET_B has also been linked to renal failure, congestive heart failure, atherosclerosis, and pulmonary hypertension (D’Orléans-Juste, et al., 2002). A mutation in ET_B has been found to cause Hirschsprung disease-2, a degenerative disease of the digestive tract (Puffenberger et al, 1994). Millipore’s cloned human ET_B expressing cell line is made in the CHO host, which supports high levels of recombinant ET_B expression on the cell surface. Thus, the ET_B Membrane Preparation is an ideal tool for screening for compounds binding to ET_B. 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 ET_B Membrane Prep yields greater than 5 fold signal-to-background ratio.

Radioligand binding assay and GTPγS binding

GPCR Class: A

Protein Target: ETB

Target Sub-Family: Endothelin

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 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.

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.

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.

Table 1. Signal:background and specific binding values obtained in a competition binding assay with ET_B membrane prep.

	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

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

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.