ChemiSCREEN Membrane Preparation Recombinant Human sst₁ Somatostatin Receptor

Human sst₁

Somatostatin, a 14 or 28 amino acid peptide, inhibits hormone secretion from exocrine glands, and displays an antiproliferative activity on a number of cell types. A family of 5 G_i-coupled receptors mediates the effects of somatostatin. The classical activity of somatostatin of inhibiting growth hormone release from the pituitary appears to be mediated by sst1. In addition, sst₁ functions as an inhibitory autoreceptor on somatostatin neurons in the hippocampus, hypothalamus, basal ganglia and retina (Thermos et al., 2006). The somatostatin receptors are targets for anticancer drugs and for treatment of hormonal dysfunction. Millipore′s sst₁ 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 sst₁. The membrane preparations exhibit a Kd of 1.4 nM for [¹²⁵I]- somatostatin-14. With 0.5 nM [¹²⁵I]- somatostatin-14, 5 μg/well sst1 Membrane Prep yields greater than 12 fold signal-to-background ratio.

Human sst1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay and GTPγS binding

GPCR Class: A

Protein Target: sst1

Target Sub-Family: Somatostatin

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of sst₁ membrane prep.

	5 µg/well
Signal:Background	20.4
Specific Binding (cpm)	14,647

SPECIFICATIONS: 1 unit = 5 μg membrane preparation
Bmax: 8.4 pmol/mg
K_d: 1.4 nM

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, 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 (PerkinElmer NEX389)
Wash Buffer: 50 mM HEPES, pH 7.4, 500 mM NaCl, 0.1% BSA, filtered and stored at 4°C.

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 12-fold signal:background with [¹²⁵I]- somatostatin-14 at 0.5 nM.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

Store at –70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.

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