ChemiSCREEN Human PK1 Prokineticin Receptor Membrane Preparation

General description

Full-length human GPR73 cDNA, encoding PK1

Prokineticins, also known as endocrine gland vascular endothelial growth factors (EG-VEGF), are two ~10 kD secreted proteins originally described to mediate angiogenesis and gastrointestinal smooth muscle contraction (Li et al., 2001; LeCouter et al., 2003). Subsequently, prokineticins have been found to mediate central nervous system functions including circadian rhythms and olfactory bulb development (Cheng et al., 2002; Ng et al., 2005). Two Gq-coupled receptors, PK1 and PK2 (also known as GPR73a and GPR73b), mediate cellular responses to prokineticins (Lin et al., 2002). Chemicon′s PK1 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 PK1 interactions and its ligands. The membrane preparations exhibit a Kd of 0.11 nM for [¹²⁵I]-Mamba Intestinal Toxin-1 (MIT-1). With 0.2 nM [¹²⁵I]-MIT-1, 2.5 µg/well and 5 µg/well PK1 Membrane Prep typically yield greater than 5-fold signal-to-background ratio.

Application

Radioligand binding assay and GTPγS binding.

Biochem/physiol Actions

GPCR Class: A

Protein Target: PK1 / PKR1

Target Sub-Family: Prokineticin

Quality

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of PK1 membrane prep:

	5 µg/well	2.5 µg/well
Signal:Background	6	6.4
Specific Binding (cpm)	3166	1798

SPECIFICATIONS: 1 unit = 5 µg
Bmax for [¹²⁵I] MIT-1 binding: 0.58 pmol/mg protein;
Kd for [¹²⁵I] MIT-1 binding: ~ 0.11 nM

Specifications

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]-MIT-1 (Perkin Elmer#:NEX-410 )
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 10-fold signal:background with ¹²⁵I-labeled MIT-1 at 0.2 nM

Physical form

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.

Storage and Stability

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

Legal Information

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

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.