ChemiSCREEN Membrane Preparation Recombinant Human 5-HT_1B Serotonin Receptor

32161000

Full-length human HTR1B cDNA encoding the 5-HT_1B Serotonin Receptor
5-Hydroxytryptamine (5-HT, also commonly known as serotonin) is synthesized in enterochromaffin cells in the intestine and in serotonergic nerve terminals. In the periphery, 5-HT mediates gastrointestinal motility, platelet aggregation, and contraction of blood vessels. Many functions of the central nervous system are influenced by 5-HT, including sleep, motor activity, sensory perception, arousal and appetite. A family of 12 GPCRs and one ion channel mediate the biological effects of 5-HT (Hoyer et al., 1994). The 5-HT_1B receptor (also known as 5-HT_1Dβ) is expressed presynaptically on serotonergic neurons and postsynaptically on non-serotonergic neurons, and it regulates release of 5-HT and acetylcholine. Several brain regions, including globus pallidus, substantia nigra and dorsal subiculum, highly express HT1B. 5-HT_1B has been implicated in the physiology of aggression, depression, migraine, anxiety and reward for drugs of abuse (Sari, 2004). 5-HT_1B interacts with p11, which increases cell surface expression and function of 5-HT_1B (Svenningsson et al., 2006). Millipore′s 5-HT_1B 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-HT_1B. The membrane preparations exhibit a Kd of 10.5 nM for [³H]-GR125743. With 7 nM [³H]-GR125743, 10μg/well 5-HT_1B Membrane Prep typically yields greater than 8-fold signal-to-background ratio.

Radioligand binding assay and GTPγS binding.

Target Sub-Family: Serotonin
GPCR Class: A
Protein Target: 5-HT1B

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of sst5 Receptor membrane prep.

	10 µg/well	20 µg/well
Signal:Background	12.4	10.5
Specific Binding (cpm)	3210	3352

SPECIFICATIONS: 1 unit =10 μg
Bmax for [³H]-GR125743 binding: 3.7 pmol/mg protein
Kd for [³H]-GR125743 binding: ~10.5 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 50mM Tris, pH 7.4. 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, 10 mM MgCl2, 1 mM EDTA, filtered and stored at 4°C

Radioligand: [³H]-GR125743. (Amersham #:TRK-1046 )

Wash Buffer: 50 mM Tris, pH 7.4, filtered and stored at 4°C.

One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 8-fold signal:background with ³H labeled GR125743 at 7 nM

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml 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_000863.1

P28222

dry ice

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

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