Application

Radioligand binding assay and GTPγS binding.

Biochem/physiol Actions

Protein Target: sst2

GPCR Class: A

Target Sub-Family: Somatostatin

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

Full-length human SSTR2 cDNA encoding sst₂

Somatostatin (sst) is a multifunctional peptide with two biologically active forms, sst-14 and sst-28, which are synthesized in neurons throughout the brain as well as in peripheral tissues such as the pancreas and the gut (Gillies, 1997). SST exerts a diverse array of effects that include inhibition of endocrine secretion, modulation of neurotransmission, and regulation of cell proliferation by stimulating a family of five G-protein-coupled receptors. Somatostatin receptor sst₂ mRNA is predominantly expressed in central nervous system. Study using sst₂ knock-out mice has found the increased anxiety-related behaviour while locomotor and exploratory activity was decreased in stress-inducing situations (coupled with an increase in pituitary ACTH release, a regulator of the stress response) (Viollet et al., 2000) . In the periphery, inhibition of glucagon release by sst in mouse islets is primarily mediated via sst₂ (Strowski et al., 2000). In addition, endogenous sst functions through sst₂ to suppress gastric acid secretion through inhibition of gastrin activity (Martinez et al., 1998). Millipore′s sst₂ 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 of sst₂. The membrane preparations exhibit a Kd of 0.95 nM for [¹²⁵I]-Somatostatin 14. With 0.75 nM [¹²⁵I]-Somatostatin 14, 5 µg/well sst₂ Membrane Prep typically yields greater than 20-fold signal-to-background ratio.

Legal Information

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

Physical form

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of sst₂ Receptor membrane prep.

Specifications

Inucbation ConditionsRECOMMENDED ASSAY 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 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]-Somatostatin 14. (Perkin Elmer NEX-389 )

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 20-fold signal:background with 125I labeled somatostatin 14 at 0.75 nM

Storage and Stability

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