ChemiSCREEN Membrane Preparation Recombinant Human 5-HT₆ Serotonin Receptor

32161000

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.

Radioligand binding assay and GTPγS binding.

Target Sub-Family: Serotonin
GPCR Class: A
Protein Target: 5-HT6

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

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

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.

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

100

human

expressed in Chem-1 cells

ChemiScreen
Chemicon^®

ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable

NM_000871

P50406

dry ice

Maintain frozen at –70°C up to expiration date indicated on the label. Do not freeze and thaw.

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