"HCAR2" Related Products

Human HCAR2/Galpha15 Stable Cell Line-Chem-4

Cat.No.: CSC-RG0708
Cell Line Description: This human GPR109A-expressing cell line is made in the Chem-4 host, which supports high levels of recombinant GPR109A expression on the cell surface and contains optimal levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between GPR109A and its ligands.
Background: Nicotinic acid (niacin), a vitamin of the B complex, is used clinically in high doses to decrease total plasma levels of cholesterol. Interestingly, the total cholesterol levels and low-density lipoprotein (LDL) concentrations decrease, while the high-density lipoprotein (HDL) concentrations increase with nicotinic acid treatment. The cholesterol-lowering effects of nicotinic acid result from inhibition of lipolysis in adipose tissue, which decreases plasma levels of free fatty acid (FFA). In a study of nicotinic acid and myocardial infarction in the Coronary Drug Project (1966-1975), patients receiving 3 g/day nicotinic acid exhibited reduced rates of myocardial infarction. However, an unwanted effect of high doses of nicotinic acid is vasodilatation, occurring mainly in the upper body and face, known as flushing. Recently two receptors for nicotinic acid have been identified as class A G protein-coupled receptors that couple to Gi to inhibit accumulation of cAMP. GPR109A (also known as HM74A in humans and PUMA-G in mice) is a high affinity receptor for nicotinic acid, whereas GPR109B (also known as HM74) is a low affinity receptor for nicotinic acid that is found in humans but not rodents. GPCR109A is found primarily in adipose tissue and immune cells.
Growth Properties: Adherent
Host Cell: Chem-4
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of HCAR2.2. Protein expression: HCAR2 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by Nicotinic Acid: ~19 nM; EC50 for calcium mobilization by Acifran: ~249 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Nicotinic
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 + 250μg/mL Hygromycin Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-Strep Atmosphere: 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: HCAR2 hydroxycarboxylic acid receptor 2 [ Homo sapiens ]
Official Symbol: HCAR2
Synonyms: HCAR2; hydroxycarboxylic acid receptor 2; G protein coupled receptor 109A , GPR109A; HCA2; HM74A; niacin receptor 1; NIACR1; Puma g; PUMAG; nicotinic acid receptor; G protein-coupled receptor 109A; G-protein coupled receptor 109A; G protein-coupled receptor HM74a; G-protein coupled receptor HM74A; hydroxy-carboxylic acid receptor 2; HM74a; HM74b; Puma-g; GPR109A;
Gene ID: 338442
mRNA Refseq: NM_177551
Protein Refseq: NP_808219
MIM: 609163
UniProt ID: Q8TDS4
Chromosome Location: 12q24.31
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class A Rhodopsin-like, organism-specific biosystem; Signal Transduction, organism-specific biosystem; Signaling by GPCR, organism-specific biosystem;
Function: G-protein coupled purinergic nucleotide receptor activity; nicotinic acid 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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