ChemiSCREEN Human NK₁ Tachykinin Receptor Membrane Preparation

Full-length human TACR1 cDNA encoding NK1

The tachykinin peptide family in mammals comprises three peptides, substance P, neurokinin A and neurokinin B, which bind to the tachykinin receptor family of GPCRs, NK1, NK2 and NK3 (Severini et al., 2002). Tachykinins have prominent activity in the GI system, in which they stimulate intestinal contraction and salivation. These effects are mediated by NK1 and NK2, and an antagonist of NK1, aprepitant, is used for treatment of chemotherapy-induced emesis (Rupniak and Kramer, 1999). The NK1 tachykinin receptor is expressed in brain, and is thought to be involved in depression and nociception (Saria, 1999). Although NK1 appears to promote nociception in animal models, the significance of this activity is controversial, as antagonists of NK1 have not proven efficacious in pain relief in humans (Hill, 2000). Chemicon′s NK1 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 NK1 interactions with Substance P. The membrane preparations exhibit a Kd of 0.28-0.4 nM for [125I]-Substance P. With 10 µg/well NK1 Membrane Prep and 0.25 nM [¹²⁵I]-Substance P, a greater than 5-fold signal-to-background ratio was obtained.

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: NK1

Target Sub-Family: Tackykinin /neurokinin

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 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]-Substance P (Perkin Elmer#:NEX190 )
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 a unit is the amount of membrane that will yield greater than 5-fold signal:background with ¹²⁵I labeled Substance P at 0.25 nM

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.

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of NK1 membrane prep:

	10 µg/well
Signal:Background	7.17
Specific Binding (cpm)	2210

SPECIFICATIONS: 1 unit = 10 µg
Bmax : 0.38 pmol/mg
Kd: 0.34 nM

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