ChemiSCREEN Membrane Preparation Recombinant Human 5-HT6 Serotonin Receptor

General description

Full-length human HTR6 cDNA encoding the 5-HT₆ Serotonin Receptor

The neurotransmitter serotonin/5-hydroxytryptamine (5-HT) regulates a wide variety of neurological functions. A family of 13 receptors (12 GPCRs and one ion channel) mediate the effects of serotonin. The serotonin receptor 5-HT₆ is a Gs coupled receptor expressed solely in the CNS, primarily in the limbic and cortical regions. 5-HT₆ appears to play a role in memory and learning, obesity, psychosis, anxiety and epilepsy (Woolley et al., 2004; Fisas et al., 2006). In particular, a 5-HT₆-selective agonist caused significant weight loss in a rat model of diet-induced obesity. Millipore′s 5-HT₆ 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 5-HT₆. The membrane preparations exhibit a Kd of 0.44 nM for [¹²⁵I]-SB258585. With 0.25nM [¹²⁵I]-SB258585, 5 μg/well 5-HT₆ Membrane Prep typically yields greater than 6-fold signal-to-background ratio.

Application

Radioligand binding assay and GTPγS binding.

Biochem/physiol Actions

GPCR Class: A

Protein Target: 5-HT6

Target Sub-Family: Serotonin

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of 5-HT₆ Receptor membrane prep.

	5 µg/well	10 µg/well
Signal:Background	12.8	12.3
Specific Binding (cpm)	10192	12675

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
B_max for [¹²⁵I]-SB258585 binding: 6.1 pmol/mg protein
K_d for [¹²⁵I]-SB258585 binding: ~0.44 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 the binding buffer. 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: 20 mM HEPES, 3 mM MgCl₂, 2 mM Ascorbic acid, pH 7.4, filtered and stored at 4°C

Radioligand: [¹²⁵I]-SB258585 (Perkin Elmer #:NEX424)

Wash Buffer: same as the binding buffer.

One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 6-fold signal:background with ¹²⁵I labeled SB258585 at 0.25 nM

Physical form

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.

Storage and Stability

Maintain frozen at –70°C up to expiration date indicated on the label. 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.