ChemiSCREEN NTR1 Membrane Preparation

Human NTR1

Neurotensin is a 13 amino acid peptide that induces hypothermia and mediates analgesia when administered centrally, and also mediates the behavioral effects of antipsychotic drugs in rodents (Kinkead and Nemeroff, 2004). In addition, several intestinal functions, such as contractility and inflammation, are mediated by neurotensin (Castagliuolo et al., 1999). The actions of neurotensin are mediated by two GPCRs, NTR1 (or NTS1) and NTR2 (or NTS2). NTR1 knockout mice are resistant to the hypothermic, analgesic and contractile effects of neurotensin, indicating that NTR1 is the primary mediator of these effects (Pettibone et al., 2002). Small molecule antagonists, including SR48692 and SR142948A, that are partially selective for NTR1 also interfere with the biological effects of neurotensin (Labbé-Jullié et al., 1998).

Chemicon′s NTR1 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 antagonists of neurotensin/NTR1 interactions. The membrane preparations exhibit a Kd of 1.5 nM for [¹²⁵I]-neurotensin. With 2.5 μg/well NTR1 Membrane Prep and 0.5 nM [¹²⁵I]-neurotensin, a greater than 7-fold signal-to-background ratio is obtained.

Radioligand binding assay, and GTPgammaS binding.

GPCR Class: A

Protein Target: NTR1 / NTS1

Target Sub-Family: Neurotensin

Inucbation Conditions
Binding buffer: 50mM HEPES, 5mM MgCl2, 1mM CaCl2, 0.2% BSA, pH to 7.4 and filter.
Radioligand: [125I] Neurotensin (PerkinElmer#: NEX198)
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 neurotensin at 0.5 nM.

PACKAGING: Packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSAPackaging method: Membranes protein were adjusted to 0.5 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.

Signal:background and specific binding values obtained in a competition binding assay with NTR1 membrane prep:

	5 µg/well
Signal:Background	47
Specific Binding (cpm)	17556

SPECIFICATIONS: 1 unit = 5 µg
Bmax: 12.9 pmol/mg
Kd: 1.5 nM

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