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General description
TRANSFECTION: Full-length human GCGR cDNA encoding Glucagon Receptor
HOST CELLS: Chem-1, an adherent mammalian cell line with no endogenous glucagon receptor expression.
Glucagon is a 29-amino acid peptide that stimulates glycogenolysis and gluconeogenesis in the liver to increase blood glucose. The receptor for glucagon is a class 2 (or class B) GPCR that signals through Gs to stimulate cAMP production (Mayo et al., 2003). Mice lacking the glucagon receptor have mild hypoglycemia after fasting, and exhibit hyperplasia of pancreatic α-cells (Gelling et al., 2003). Because of its role in promoting hyperglycemia, the glucagon receptor presents a potential target for treatment of diabetes. Chemicon′s glucagon receptor 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 glucagon receptor interactions and its ligands. The membrane preparations exhibit a Kd of 3.09 nM for [125I]-glucagon. With 1 nM [125I]-glucagon, 5 or 10 μg/well glucagon receptor Membrane Prep yields greater than 10-fold signal-to-background ratio.
Features and Benefits
Inucbation Conditions
RECOMMENDED ASSAY CONDITIONS: Membranes are mixed with radioactive ligand and unlabeled competitor 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 Hepes, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA, filtered and stored at 4°C
• Radioligand: [125I]-glucagon (Perkin Elmer#:NEX-207)
• Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, 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 10-fold signal:background with [125I]-labeled glucagon at 1 nM
Physical form
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA; no preservatives.
Packaging method: Membrane proteins were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.
Analysis Note
SPECIFICATIONS:
1 unit = 5 μg
Bmax for [125I] glucagon binding: 16.7 pmol/mg protein
Kd for [125I] glucagon binding: ~ 3.09 nM
Signal:background and specific binding values obtained in a competition binding assay with varying amounts of Glucagon receptor membrane prep:
| 10 µg/well | 5 µg/well | |
|---|---|---|
| Signal:Background | 10.1 | 35 |
| Specific Binding (cpm) | 33315 | 36252 |
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
| biological source | human |
| Quality Segment | 100 |
| 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. | NM_000160.2 |
| UniProt accession no. | P47871 |
| shipped in | dry ice |
| storage temp. | -65 to -96°C |
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