Application

Radioligand binding assay and GTPγS binding

Biochem/physiol Actions

Protein Target: GABAB1b

Target Sub-Family: GABAB

GPCR Class: C

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.

General description

The neurotransmitter γ-aminobutyric acid (GABA) exerts its effects through an ion channel, GABA_A, and a GPCR, GABA_B. Functional GABA_B is a heterodimer composed of the GABA_B1 and GABA_B2 subunits, which share 35% sequence identity and belong to the class 3 family of GPCRs. The GABA_B1 subunit, which exists as splice variants GABA_B1a and GABA_B1b, binds directly to GABA and is required for agonist activation. The GABA_B2 and GABA_B1 subunits associate by formation of a coiled coil by their C-terminal tails; this association masks an ER retention sequence in GABA_B1 to permit export from the ER and trafficking to the cell surface. In addition to its chaperone function, GABA_B2 is the component that couples to Gi to reduce intracellular cAMP. Agonists of GABA_B, such as baclofen, are used clinically for treatment of muscle spasticity, migraine headache and musculoskeletal pain (Bowery et al., 2002). Chemicon′s GABA_B1b 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 GABA_B1b receptor interactions with its ligand. The membrane preparations exhibit a Kd of 1.15nM for [³H]-CGP 54626A. With 5.0 ug/well GABA_B1b Membrane Prep and 7.5nM [³H]-CGP 54626A, a greater than 5-fold signal-to-background ratio was obtained.

Full-length human GABBR1b and GPR51 cDNAs encoding GABA_B1b/GABA_B2

Legal Information

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

Physical form

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with 5ug/well of GABA_B1b Receptor membrane prep.

Specifications

Inucbation ConditionsMembranes 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: 1X Hanks′ Balanced Salt Solution (HBSS): GIBCO Cat. No. 14175
Radioligand: [³H]-CGP 54626A (American Radiolabeled Chemicals Inc., ART 517)
Wash Buffer: 1X Hanks′ Balanced Salt Solution (HBSS): GIBCO Cat. No. 14175

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 5-fold signal:background with ³H-labeled CGP 54626A at 7.5nM.

Storage and Stability

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