HTS139M

ChemiSCREEN Membrane Preparation Recombinant Human sst5 Somatostatin Receptor

Human sst5 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

• UNSPSC Code: 41106514
• eCl@ss: 32161000
• NACRES: NA.84

Properties

• biological source: human
• Quality Level: 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_001053.1
• UniProt accession no.: P35346
• shipped in: dry ice

Description

General description

Full-length human SSTR5 cDNA encoding sst5

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 sst₅ is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. In mice, sst5 mediates inhibition by somatostatin of pancreatic insulin secretion and contributes to the regulation of glucose homeostasis and insulin sensitivity (Strowski et al. 2003). In addition, deficiency of sst5 leads to subtype-selective sexually dimorphic changes in the expression of both brain and pancreatic somatostatins (Ramirez et al. 2004). Millipore′s sst5 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 sst5. The membrane preparations exhibit a Kd of 1.2 nM for [¹²⁵I]-Somatostatin 14. With 0.5 nM [¹²⁵I]-Somatostatin 14, 5 μg/well sst5 Membrane Prep typically yields greater than 10-fold signal-to-background ratio.

Application

Human sst5 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay and GTPγS binding.

Biochem/physiol Actions

GPCR Class: A

Protein Target: sst5

Target Sub-Family: Somatostatin

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of sst5 Receptor membrane prep.

	10 µg/well	5 µg/well
Signal:Background	15.2	15.7
Specific Binding (cpm)	25710	21425

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
B_max for [¹²⁵I]-Somatostatin binding: 10.4 pmol/mg protein
K_d for [¹²⁵I]-Somatostatin binding: ~1.2 nM

Specifications

Inucbation Conditions
RECOMMENDED 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 10-fold signal:background with ¹²⁵I labeled somatostatin 14 at 0.5 nM.

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

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.

Safety Information

• Storage Class Code: 10 - Combustible liquids
• WGK: WGK 2