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Human QRFPR/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0728
Cell Line Description: This human GPR103-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant GPR103 expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to enhance coupling the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between GPR103 and its ligands.
Background: GPR103 is a Gq-coupled receptor that is located in the brain and adrenal gland, and is activated by a 43-amino acid RFamide peptide, QRFP43, and a 26 amino acid fragment, QRFP26/26RFa/Peptide 518. QRFP43 is the active form in rat brain, and intracerebral administration of QRFP43 induces feeding and metabolic rate.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of QRFPR.2. Protein expression: QRFPR in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by human 26RFa: ~ 0.64 nM.
Application: Calcium flux assay, radioligand bindiing assay
Sub-type: Orexin
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: QRFPR pyroglutamylated RFamide peptide receptor [ Homo sapiens ]
Official Symbol: QRFPR
Synonyms: QRFPR; pyroglutamylated RFamide peptide receptor; G protein coupled receptor 103 , GPR103; QRFP receptor; G protein-coupled receptor 103; G-protein coupled receptor 103; orexigenic neuropeptide QRFP receptor; AQ27; GPR103; SP9155; MGC149217;
Gene ID: 84109
mRNA Refseq: NM_198179
Protein Refseq: NP_937822
MIM: 606925
UniProt ID: Q96P65
Chromosome Location: 4q27
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; Orexin and neuropeptides FF and QRFP bind to their respective receptors, organism-specific biosystem; Peptide ligand-binding receptors, organism-specific biosystem; Signal Transduction, organism-specific biosystem;
Function: G-protein coupled receptor activity; neuropeptide Y receptor activity; receptor activity; signal transducer activity;

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Optional requirements on this protein    +Expand
C-fusion    N-fusion   Non-tagged
His    GST   Fc   Others
<1.0 eu/μg    <0.1 eu/μg   <0.01 eu/μg   Not required
Monomer Isolation    Dimer Isolation    Not required
>80% by SDS-PAGE    >90% by SDS-PAGE   >95% by SDS-PAGE   Others

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