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Human ADRA2A/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0607
Cell Line Description: This human alpha2A-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant alpha2A 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-alpha2A and its ligands.
Background: The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the alpha- and beta-adrenoceptors. The alpha2 adrenergic receptor subfamily members, consisting of alpha2A, alpha2B, and alpha2C, couple primarily to Gi to inhibit cAMP production, and play an important role in regulation of cardiovascular and CNS function. The alpha2A receptor at presynaptic sites has an inhibitory effect on catecholamine release from sympathetic nerve endings. Experiments with alpha2A-selective agonists and mice lacking alpha2A demonstrate that activation of alpha2A results in hypotension, sedation, analgesia, and hypothermia.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of ADRA2A.2. Protein expression: ADRA2A in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by UK14,304: ~ 2.3 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Adrenoceptors
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: ADRA2A adrenoceptor alpha 2A [ Homo sapiens ]
Official Symbol: ADRA2A
Synonyms: ADRA2A; ADRA2; ADRA2R; ADRAR; ALPHA2AAR; ZNF32; adrenoceptor alpha 2A; alpha-2A adrenergic receptor; alpha-2A adrenoceptor; alpha-2AAR subtype C10; adrenergic, alpha-2A-, receptor; alpha-2 adrenergic receptor subtype C10; alpha-2-adrenergic receptor, platelet type
Gene ID: 150
mRNA Refseq: NM_000681.3
Protein Refseq: NP_000672.3
MIM: 104210
UniProt ID: P08913
Chromosome Location: 10q25.2
Pathway: Adrenaline signalling through Alpha-2 adrenergic receptor; Adrenoceptors; Amine ligand-binding receptors; Class A/1 (Rhodopsin-like receptors); G alpha (i) signalling events; G alpha (z) signalling events; GPCR downstream signaling;
Function: alpha-1B adrenergic receptor binding; alpha-2C adrenergic receptor binding; alpha2-adrenergic receptor activity; epinephrine binding; heterotrimeric G-protein binding; norepinephrine binding; protein binding; protein heterodimerization activity; protein homodimerization activity; protein kinase binding; thioesterase binding

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