ChemiScreen VPAC₁ Membrane Preparation

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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 50mM 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] VIP (Perkin Elmer# NEX192)

Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, 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 5-fold signal:background with 125I-labeled VIP at 0.4 nM

Human VIPR1 cDNA, encoding VPAC1

Vasoactive intestinal peptide (VIP), a 28 amino acid peptide originally isolated by its vasodilation activity, binds to two class B GPCRs, VPAC1 and VPAC2, to exert its functions in the CNS, vasculature, immune system and adrenal medulla (Harmar et al., 1998). In the immune system, VIP is synthesized by mast cells and lymphocytes, and appears to inhibit inflammation and to shift the immune response toward a Th2 pathway (Delgado et al., 2004). In the heart, VIP is expressed by nerve fibers, where it modulates heart rate, and coronary blood flow (Henning and Sawmiller, 2001). Chemicon′s VPAC1 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 VPAC1 interactions with VIP. The membrane preparations exhibit a Kd of 1.2 nM for [125I]-VIP. With 2.5 μg/well VPAC1 Membrane Prep and 0.4 nM [125I]-VIP, a greater than 5-fold signal-to-background ratio is obtained.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA 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.

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

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of VPAC₁ membrane prep:

	10 µg/well	5 µg/well
Signal:Background	9.7	10.8
Specific Binding (cpm)	30299	31224

SPECIFICATIONS:
1 unit = 5 µg
Bmax: 19.65 pmol/mg
Kd: 1.2 nM

Radioligand binding assay, and GTPgammaS binding.

GPCR Class: B

Protein Target: VPAC1 / VIP1

Target Sub-Family: VIP/PACAP