ChemiSCREEN™ Membrane Preparation Recombinant Human GIP Glucagon Family Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human GIP Glucagon Family Receptor

• ChemiScreen
• Chemicon (Millipore)

Full-length human GIPR cDNA encoding GIP Receptor

Gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic polypeptide, is a 42 amino acid peptide that is secreted from the duodenum and proximal jejunum after food intake. The receptor for GIP is a family 2 GPCR that is most closely related to the glucagon and GLP-1 receptors, and like these receptors it couples to Gs to stimulate intracellular cAMP (Mayo et al., 2003). The GIP receptor is most highly expressed in pancreas, and the primary physiological function of GIP is to potentiate glucose-mediated secretion of insulin (Yip et al., 1999). The GIP receptor is expressed in many other tissues, and accordingly GIP also has extrapancreatic activities. GIP secreted after meal ingestion stimulates bone formation, and mice lacking the GIP receptor display features of osteoporosis (Tsukiyama et al., 2006). In addition, GIP increases insulin sensitivity in adipose tissue. Since patients with type 2 diabetes display decreased responsiveness to GIP, a defect in GIP receptor expression and/or signaling may lead to β-cell dysfunction and type 2 diabetes (Meier and Nauck, 2004). Millipore's GIP 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 agonists and antagonists at GIP Receptor. The membrane preparations exhibit a Kd of 0.44 nM for [¹²⁵I]-GIP. With 0.2 nM [¹²⁵I]-GIP, 10 µg/well GIP Receptor Membrane Prep typically yields greater than 8-fold signal-to-background ratio.

Radioligand binding assay

Human

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GPCR

GIPR, Gastric inhibitory polypeptide

Chem-9

• GIPR
• MGC126722
• GIP-R

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 the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

Table 1. Signal:background and specific binding values obtained in a competition binding assay with GIP Receptor membrane prep and unlabeled GIP.

	10 µg/well
Signal:Background	12.7
Specific Binding (cpm)	11304

SPECIFICATIONS: 1 unit = 10 µg
Bmax for [¹²⁵I] GIP binding: 3.7 pmol/mg protein
Kd for [¹²⁵I] GIP binding: ~0.44 nM

GPCR Membrane Preparations

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 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] GIP (Perkin Elmer#: NEX-402)
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 8-fold signal:background with ¹²⁵I-labeled GIP at 0.2 nM

Store at –80°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.

Human