ChemiSCREEN Membrane Preparation Recombinant Human ET_B Endothelin Receptor

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

Radioligand binding assay and GTPγS binding

GPCR Class: A

Protein Target: ETB

Target Sub-Family: Endothelin

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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.

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.

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.

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