CCK1 Membrane Preparation

Application

Radioligand binding assay and GTPγS binding

Biochem/physiol Actions

Protein Target: CCK1 / CCKa

Target Sub-Family: Cholecystokinin

GPCR Class: A

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.

General description

Human CCK₁ Receptor

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. CCK₁ selectively binds sulfated CCK, whereas CCK₂ binds to CCK and gastrin with similar affinity. Binding of ligands to CCK₁ stimulates mobilization of intracellular calcium by activation of G_q/11. CCK₁ receptors in the periphery are primarily localized in the pancreas, gallbladder, pylorus, intestine where they are responsible for the regulation of diverse digestive processes. They are also present in select areas of the peripheral nervous system (vagus nerve), and the CNS where they mediate the satiety effects of CCK, regulate an increase in dopamine release, and antagonize opioid analgesia (Noble et al., 1999). Chemicon′s CCK₁ 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 CCK₁ interactions and its ligands. The membrane preparations exhibit a Kd of 0.5 nM for [¹²⁵I]-CCK-8 sulfated. With 10.0ug/well CCK1 membrane prep and 0.5 nM [¹²⁵I]-CCK-8 sulfated, a greater than 4-fold signal-to-background ratio was obtained.

Legal Information

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

Physical form

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 4-fold signal:background with ¹²⁵I-labeled CCK-8 at 0.5 nM.

Liquid in packaging buffer: 50 mM Tris, pH 7.4, 10% glycerol with no preservatives.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of CCK₁ Receptor membrane prep.

Specifications

Inucbation ConditionsMembranes 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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) is blocked 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.
Note: Due to the acidic property of the labeled sulfated CCK-8, filter plates coated with PEI bind the labeled ligand nonspecifically and result in elevated backgrounds.

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] -CCK-8 sulfated (Perkin Elmer # NEX203)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl, 0.1% BSA, filtered and stored at 4°C.

Storage and Stability

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