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HTS083M

Sigma-Aldrich

ChemiScreen Recombinant Human CCK2 Cholecystokinin Receptor Membrane Preparation

Human CCK2 / CCKb GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

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eCl@ss:
32161000
NACRES:
NA.84

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

Gene Information

human ... CCKBR(887)

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HTS184MHTS080MHTS115M
recombinant

expressed in Chem-1 cells

recombinant

expressed in Chem-1 cells

recombinant

expressed in Chem-1 cells

recombinant

expressed in Chem-1 cells

manufacturer/tradename

ChemiScreen

manufacturer/tradename

ChemiScreen, Chemicon®

manufacturer/tradename

ChemiScreen, Chemicon®

manufacturer/tradename

ChemiScreen, Chemicon®

technique(s)

ligand binding assay: suitable (GTPγS)

technique(s)

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

technique(s)

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

technique(s)

radioligand binding assay (RLBA): suitable

NCBI accession no.

NM_176875.2

NCBI accession no.

NM_000730.2

NCBI accession no.

NM_001058.2, NM_015727.1

NCBI accession no.

NM_000739.2, NM_001006626.1, NM_001006627.1, NM_001006628.1, NM_001006629.1, NM_001006630.1, NM_001006631.1, NM_001006632.1

UniProt accession no.

P32239

UniProt accession no.

P32238

UniProt accession no.

P25103

UniProt accession no.

P08172

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. 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 Gq/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

Application

Radioligand binding assay and GTPγS binding.

Biochem/physiol Actions

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

Quality

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of CCK2 membrane prep:
10 µg/well5 µg/well
Signal:Background 27.4 27.8
Specific Binding (cpm) 25,302 20,111


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

Physical form

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.

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 2

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable


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