ChemiSCREEN Membrane Preparation Recombinant Human 5-HT₇ Serotonin Receptor

General description

Full-length human HTR7 cDNA encoding the 5-HT₇ 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-HT₇ is a Gs coupled receptor expressed in the brain (hypothalamus, hippocampus and frontal cortex) and in smooth muscle and GI tract. Systemic delivery of serotonin results in hypothermia, and pharmacological and genetic studies indicate that 5-HT₇ mediates this effect (Hedlund et al., 2003). In addition, 5-HT₇ appears to modulate circadian rhythms and smooth muscle relaxation, and is implicated as a target for sleep, particularly as related to depression, and migraine (Terron and Falcon-Neri, 1999; Mullins et al., 1999). Millipore′s 5-HT₇ 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-HT₇. The membrane preparations exhibit a Kd of 37.7 nM for [³H]-5-Carboxamidotryptamine (5-CT). With 30 nM [³H]-5-CT, 10 μg/well 5-HT₇ Membrane Prep typically yields greater than 4-fold signal-to-background ratio.

Application

Human 5-HT7 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Biochem/physiol Actions

Protein Target: 5-HT7

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with 10 μg of 5-HT₇ Receptor membrane prep.

	10 µg/well
Signal:Background	7.5
Specific Binding (cpm)	545

SPECIFICATIONS: 1 unit =10 μg
Bmax for [³H]-5-CT binding: 20.1 pmol/mg protein
Kd for [³H]-5-CT binding: ~37.7 nM

Specifications

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 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: 50 mM Tris, pH 7.4, 5 mM MgCl₂, 4 mM CaCl₂, 0.5 mM ascorbic acid, filtered and stored at 4°C

Radioligand: [³H]-5-Carboxamidotryptamine (Perkin Elmer NET1071)

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 4-fold signal:background with ³H-labeled 5-CT at 30 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 for up to 2 years. Do not freeze and thaw.

Legal Information

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