Description
General description
Human 5-HT5A
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 (Hoyer et al., 1994). The serotonin receptors 5-HT5A and 5-HT5B are Gi/o coupled receptors mainly expressed in the CNS but not in peripheral organs. 5-HT5A is found in rodents and humans and is broadly expressed in the CNS whereas 5-HT5B exists in rodents, but not humans, and is expressed in a more restricted fashion (Grailhe et al., 2001). The hallucinogenic drug LSD functions as a partial agonist at 5-HT5A, and mice lacking 5-HT5A display reduced LSD-induced locomotion (Nelson 2004). HT5A receptors may also be involved in a neuronally-driven mechanism for regulating astrocyte physiology, with relevance to gliosis (Glennon et al., 2000).
Millipore′s 5-HT5A 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 antagonists of 5-HT5A interactions and its ligands. The membrane preparations exhibit a Kd of 67.7 nM for [3H]-5-CT (Carboxamidotryptamine). With 30 nM [3H]- 5-CT, 10 μg/well 5-HT5A Membrane Prep yields greater than 3-fold signal-to-background ratio.
Application
Radioligand binding assay and GTPγS binding
Physical form
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.
Preparation Note
Store at -70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.
Analysis Note
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 HEPES, 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 Hepes, 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: 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 4 mM CaCl₂, 0.5 mM Ascorbic Acid, filtered and stored at 4°C.
One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 3-fold signal:background with 3H-labeled 5-carboxamidotryptamine at 30.0 nM.
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.
