Full-length human EDNRA cDNA encoding ETA

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. ETA is the predominant receptor on smooth muscle cells and thus is the primary mediator of the vasoconstrictor activity of endothelins in vivo. Endothelin receptors are also found in the epithelium and central nervous system (Davenport, 2002). Chemicon′s ETA 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 ETA interactions with its ligands. The membrane preparations exhibit a Kd of 85 pM for [¹²⁵I]-Endothelin-1. With 2.5 or 10 ug/well ETA Membrane Prep and 0.35 nM [¹²⁵I]-Endothelin-1, greater than 7-fold and 9-fold signal-to-background ratio were obtained, respectively.

Protein Target: ETA

Inucbation 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, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50 mM 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#:NEX-259 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM 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 ¹²⁵I-labeled Endothelin-1 at 0.35 nM

Bmax: 1.23 pmol/mg
Kd: 85 pM

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