Description
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
Features and Benefits
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.
Preparation Note
Maintain frozen at –70°C up to expiration date indicated on the label. Do not freeze and thaw.
Analysis Note
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
Bmax for [125I]-SB258585 binding: 6.1 pmol/mg protein
Kd for [125I]-SB258585 binding: ~0.44 nM
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.
