ChemiSCREEN GnRH Membrane Preparation

Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH), regulates the reproductive hormonal cascade in vertebrates. Upon release from the hypothalamus, GnRH stimulates secretion of luteinizing hormone and follicle-stimulating hormone from the pituitary. In humans, the type I GnRH receptor is a GPCR that is unusual in the lack of a cytoplasmic C-terminal tail. Although some species contain a type II GnRH receptor, the human version of this second receptor appears to contain mutations that render it inactive. GnRH analogs (agonists) are used in low doses in a pulsatile fashion in the treatment of infertility, delayed puberty and cryptorchidism. GnRH agonists at high doses desensitize the receptor, and are used along with antagonists in the treatment of hormone-dependent diseases (Millar et al., 2004). Chemicon′s cloned human type I GnRH receptor-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant GnRH receptor expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists of the GnRH receptor.

Human Type I GnRH receptor

Radioligand binding assay, and GTPgammaS binding.

GPCR Class: A

Protein Target: GnRH / LHRH

Target Sub-Family: GnRH

SPECIFICATIONS: Bmax: 1.0 pmol/mg
Kd: 0.3 nM

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]-[D-Trp6]-LHRH (Perkin Elmer # NEX365)

Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM NaCl , 0.1% BSA, filtered and stored at 4°C.

One vial contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 3-fold signal:background with 125I-labeled [D-Trp6]-LHRH at 0.5nM.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.Packaging method: Membranes protein were adjusted to 2.0 mg/ml in 1 mL packaging buffer, rapidly frozen, and stored at -80oC.

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

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