ChemiSCREEN Human VPAC₂ VIP Receptor Membrane Preparation

32161000

NA.84

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.
Human VPAC2

Radioligand binding assay, and GTPγS binding.

Target Sub-Family: VIP/PACAP
GPCR Class: B
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.

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

100

human

expressed in Chem-1 cells

ChemiScreen
Chemicon^®

ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable

NM_003382.3

P41587

dry ice

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.

12 - Non Combustible Liquids

WGK 2