ChemiSCREEN PTH Membrane Preparation

Human PTH1 GPCR membrane preparation for Radioligand binding Assays & GTPgammaS binding.

Radioligand binding assay, and GTPgammaS binding.

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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, a GF/C 96-well filter plate 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] PTH (Perkin Elmer#: NEX397)

Wash Buffer: 50 mM Hepes, pH 7.4, 500mM 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 5-fold signal:background with 125I-labeled PTH at 0.75 nM

Full-length human PTH1R cDNA

Parathyroid hormone (PTH) plays a critical role in mineral ion homeostasis, particularly in bone and kidney (Mannstadt et al., 1999; Gensure et al., 2005). A related peptide, parathyroid hormone-related peptide (PTHrP), plays an important role in skeletal development. Both peptides exert their biological actions by binding to a class B GPCR, PTH1R, that signals primarily by activation of adenylate cyclase (Schipani et al., 1993). Mutations in PTH1R have been identified as the cause of Jansen′s metaphyseal chondrodysplasia, Blomstrand′s chondrodysplasia, and enchondromatosis (Schipani and Provot, 2003). Intermittant treatment with PTH has important clinical utility in building bone mass in patients with osteoporosis (Rosen, 2003). Chemicon′s cloned human PTH1R-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant PTH1R 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 antagonists of interactions between PTH1R and its ligands.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.

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

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

SPECIFICATIONS: Bmax: 2.66 pmol/mg
Kd: 0.75 nM