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Human ADCYAP1R1/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0771
Cell Line Description: This human PAC1 -long-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant PAC1-long expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between the PAC1-long and its ligands.
Background: 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. 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.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of ADCYAP1R1.2. Protein expression: ADCYAP1R1 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by PACAP: ~ 0.11 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Vasoactive Intestinal Peptide
Propagation: Complete growth medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x Nonessential amino acids + 10mM HEPES + 1x Pen-Strep + 250μg/mL Genetecin/G-418 Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-StrepAtmosphere: air, 95%; carbon dioxide (CO2), 5%Temperature: 37 °C
Starting Cells From Frozen Cell Stock: 1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years. 2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37°C with 5% CO2. 3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
Subculturing: 1. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca2+ and Mg2+ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Growth Media per 1 mL trypsin. 2. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Freezing medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 20% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-Strep + 10% DMSO
Storage: Liquid nitrogen
Preservation: 1. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Subculture-Step 1). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10^6 cells/mL in Freezing Media (cell densities of 2-10 x 10^6 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen. 2. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Plating Media for plating for calcium assay.
Safety Considerations: The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach.
Ship: Dry ice
Gene Name: ADCYAP1R1 adenylate cyclase activating polypeptide 1 (pituitary) receptor type I [ Homo sapiens ]
Official Symbol: ADCYAP1R1
Synonyms: ADCYAP1R1; adenylate cyclase activating polypeptide 1 (pituitary) receptor type I; pituitary adenylate cyclase-activating polypeptide type I receptor; PAC1; PAC1R; PACAP receptor 1; PACAPR; PACAP-R1; PACAP type I receptor; pituitary adenylate cyclase activating polypeptide 1 receptor type I Hiphop; PACAPRI;
Gene ID: 117
mRNA Refseq: NM_001118
Protein Refseq: NP_001109
MIM: 102981
UniProt ID: P41586
Chromosome Location: 7
Pathway: Activation of TRKA receptors, organism-specific biosystem; Class B/2 (Secretin family receptors), organism-specific biosystem; G alpha (s) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class B Secretin-like, organism-specific biosystem; Glucagon-type ligand receptors, organism-specific biosystem;
Function: ADP-ribosylation factor binding; adenylate cyclase binding; neuropeptide binding; pituitary adenylate cyclase-activating polypeptide receptor activity; receptor activity; signal transducer activity; vasoactive intestinal polypeptide receptor activity;

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