ChemiSCREEN Human PAC₁-long Receptor Membrane Preparation

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of PAC1-long membrane prep:

	10 µg/well	5 µg/well
Signal:Background	22.9	29
Specific Binding (cpm)	63650	62987

SPECIFICATIONS:
1 unit = 5 µg membrane preparation
Bmax: 52.0 pmol/mg
Kd: 2.7 nM

Radioligand binding assay, and GTPγS binding.

GPCR Class: B

Protein Target: PAC1 long isoform / PACAP

Target Sub-Family: VIP/PACAP

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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] 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 20-fold signal:background with ¹²⁵I-labeled PACAP27 at 0.75 nM.

Human ADCYAP1R1 cDNA encoding the long isoform of PAC₁

PACAP (pituitary adenylyl cyclase-activating peptide) is a peptide that exists in 2 forms, 27 or 38 amino acids, and is related to vasoactive intestinal peptide (VIP). Three related class B GPCRs, PAC₁, VPAC₁ and VPAC₂, bind to PACAP; however, VPAC₁ and VPAC₂ have a much higher affinity for VIP than does PAC₁ (Vaudry et al., 2000). Several splice variants of PAC₁ result in proteins that differ at the N-terminus and third intracellular loop; these variants differ in their affinities for PACAP and abilities to activate G_q and G_s. High expression of PAC₁ is observed in the CNS and the adrenal medulla. Studies with PAC₁-null mice indicate that PAC₁ plays important roles in regulation of circadian rhythms, neutrophil migration, and pulmonary vascular tone (Hannibal et al., 2001; Martinez et al., 2005; Otto et al., 2004). Chemicon′s PAC₁-long 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 PAC₁-long interactions with PACAP27. The membrane preparations exhibit a Kd of 2.7 nM for [¹²⁵I]-PACAP27. With 5 µg/well PAC₁-long Membrane Prep and 0.75 nM [¹²⁵]-PACAP27, a greater than 20-fold signal-to-background ratio was obtained.

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.

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