HTS184M
ChemiSCREEN Membrane Preparation Recombinant Human CCK1 Cholecystokinin Receptor
Human CCK1 / CCKa GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
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About This Item
UNSPSC Code:
41106514
eCl@ss:
32161000
NACRES:
NA.41
biological source
human
Quality Level
recombinant
expressed in Chem-1 cells
manufacturer/tradename
ChemiScreen
Chemicon®
technique(s)
ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable
NCBI accession no.
UniProt accession no.
shipped in
dry ice
General description
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 (CCKA) and CCK2 (CCKB), 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 Gq/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.
Human CCK1 Receptor
Application
Radioligand binding assay and GTPγS binding
Biochem/physiol Actions
GPCR Class: A
Protein Target: CCK1 / CCKa
Target Sub-Family: Cholecystokinin
Quality
Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of CCK1 Receptor membrane prep.
SPECIFICATIONS: 1 unit = 10 µg membrane preparation
Bmax: 0.15 pmol/mg
Kd: 0.5 nM
| 10 µg/well | 5 µg/well | |
|---|---|---|
| Signal:Background | 5.31 | 6.16 |
| Specific Binding (cpm) | 1274.9 | 1691.4 |
Bmax: 0.15 pmol/mg
Kd: 0.5 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, 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.
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, 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.
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.
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.
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.
Storage Class Code
12 - Non Combustible Liquids
WGK
WGK 1
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Certificates of Analysis (COA)
Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.
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