ChemiScreen Recombinant Human CCK₂ Cholecystokinin Receptor Membrane Preparation

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of CCK₂ membrane prep:

	10 µg/well	5 µg/well
Signal:Background	27.4	27.8
Specific Binding (cpm)	25,302	20,111

Specifications:
1unit = 5 μg
B_max for [¹²⁵I] CCK-8 binding: 3.47 pmol/mg protein
K_d for [¹²⁵I] CCK-8 binding: ~0.47 nM

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: CCK2 / CCKb

Target Sub-Family: Cholecystokinin

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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 Tris, pH 7.4, 10 mM MgCl2, 1 mM EDTA, filtered and stored at 4°C

• Radioligand: [¹²⁵I]-CCK-8 (Perkin Elmer#:NEX-203 )

• Wash Buffer: 50 mM Tris, pH 7.4 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 20-fold signal:background with ¹²⁵I labeled CCK-8 at 0.3 nM

Cholecystokinins are a series of peptides of heterogeneous length (5 to 58 amino acids) that are derived from preprocholecystokinin and are found in gastrointestinal tissues and the central nervous system. Gastrin is a related peptide with 5 C-terminal amino acids identical to those of cholecystokinin. Two GPCRs, CCK₁ (CCK_A) and CCK₂ (CCK_B), bind to CCK and/or gastrin to mediate the biological effects of the peptides. CCK₂ binds to CCK and gastrin with similar affinity, whereas CCK₁ selectively binds sulfated CCK. Binding of ligands to CCK₂ stimulates mobilization of intracellular calcium by activation of G_q/11. In the periphery, CCK₂ is present in the stomach, where it mediates gastrin-stimulated gastric acid secretion. In the CNS, CCK₂ has been implicated in anxiety, depression, schizophrenia, depression and opioid analgesia (Noble et al., 1999). Chemicon′s CCK₂ 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 CCK2 interactions and its ligands. The membrane preparations exhibit a Kd of 0.47 nM for [¹²⁵I]-cholecystokinin octapeptide (CCK-8). With 0.3 nM [¹²⁵I]CCK-8, 5 or 10 μg/well CCK₂ Membrane Prep yields greater than 20-fold signal-to-background ratio.

Full-length human CCKB cDNA encoding CCK₂

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with 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.

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