Description
General description
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 Gq and Gs. 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.
Application
Radioligand binding assay, and GTPγS binding.
Biochem/physiol Actions
GPCR Class: B
Protein Target: PAC1 long isoform / PACAP
Target Sub-Family: VIP/PACAP
Features and Benefits
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.
Physical form
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.
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
Signal:background and specific binding values obtained in a competition binding assay with varying amounts of PAC1-long membrane prep:
10 µg/well5 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
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
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.
