ChemiSCREEN Membrane Preparation Recombinant Human GAL₁ Receptor

Galanin is a 29-30 amino acid peptide originally purified from intestine but later found to be abundant in the CNS. A family of 3 GPCRs, GAL1, GAL2 and GAL3, bind to galanin and mediate its biological effects. Galanin and its receptors have been implicated in pain, cognition, seizure activity, depression and drug addiction (Hokfelt, 2005). In particular, GAL1 mediates the anticonvulsant activity of galanin; mice lacking GAL1 are affected by spontaneous seizures, and have increased susceptibility to seizures induced by lithium-pilocarpine treatment and electrical perforant path stimulation (McColl et al., 2006; Mazarati et al., 2004). Chemicon′s GAL1 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 GAL1 interactions with galanin. The membrane preparations exhibit a Kd of 1.7 nM for [¹²⁵I]-galanin. With 5 ug/well GAL1 Membrane Prep and 0.25 nM [¹²⁵I]-galanin, a greater than 50-fold signal-to-background ratio was obtained.

Human GAL1

Radioligand binding assay, and GTPγS binding.

GPCR Class: A

Protein Target: GAL1

Target Sub-Family: Galanin

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, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50 mM HEPES, pH 7.4, 0.5% BSA. 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, 1 mM CaCl₂, 0.2% BSA, filtered and stored at 4°C Radioligand: [¹²⁵I] galanin (Perkin Elmer # NEX333)

Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.

1 mL per vial

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 50-fold signal:background with ¹²⁵I-labeled galanin 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.

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

SPECIFICATIONS: 1 unit = 5 ug membrane preparation Bmax: 19.2 pmol/mg Kd: 1.7 nM

	10 µg/well	5 µg/well
Signal:Background	77.6	75.6
Specific Binding (cpm)	38414	37384

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