ChemiScreen Recombinant Human CCK₂ Cholecystokinin Receptor Membrane Preparation

32161000

NA.84

Full-length human CCKB cDNA encoding CCK₂
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.

Radioligand binding assay and GTPγS binding.

Protein Target: CCK2 / CCKb
GPCR Class: A
Target Sub-Family: Cholecystokinin

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

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

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.

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

100

human

expressed in Chem-1 cells

ChemiScreen
Chemicon^®

ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable

NM_176875.2

P32239

dry ice

human ... CCKBR(887)

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

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.

12 - Non Combustible Liquids

WGK 2