Human VPAC2

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, VPAC₂ is expressed on stimulated CD4 T cells, and binding of T cell-derived VIP to VPAC₂ induces a shift toward the Th2 pathway (Delgado et al., 2004; Voice et al., 2004). In addition, VPAC2 is an essential regulator of the circadian pacemaker of the hypothalamic suprachiasmatic nuclei (Hughes et al., 2004). Chemicon′s VPAC₂ 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 VPAC₂ interactions with PACAP. The membrane preparations exhibit a Kd of 0.9 nM for [¹²⁵I]-PACAP27. With 5 µg/well VPAC₂ Membrane Prep and 0.5 nM [¹²⁵I]-PACAP27, a greater than 15-fold signal-to-background ratio was obtained.

Radioligand binding assay, and GTPγS binding.

Protein Target: VPAC2 / VIP2

Signal:background and specific binding values obtained in a competition binding assay with 5ug/well of VPAC2 membrane prep:

	10 µg/well	5 µg/well
Signal:Background	25.5.	22.8
Specific Binding (cpm)	32913	27650

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax: 17.3 pmol/mg
Kd: 0.9 nM

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] PACAP27 (Perkin Elmer # NEX294)
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 an unit is the amount of membrane that will yield greater than 15-fold signal:background with ¹²⁵I-labeled PACAP27 at 0.5 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.

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