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Human OPRM1/Galpha15 Stable Cell Line-Chem-5

Cat.No.: CSC-RG0711
Cell Line Description: This human mu-expressing cell line is made in the Chem-5 host, which supports high levels of recombinant mu expression on the cell surface and contains high levels of the promiscuous G protein to enhance coupling of the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between mu and its ligands.
Background: Opiates derived from the opium poppy, Papaver somniferum, have been used in for millenia for their anti-diarrheal, analgesic and euphoric properties. More recently, endogenous peptides, enkephalins, dynorphins, and endorphins, were found to bind to the same sites as opiate alkaloids. The receptors for the classical opioids are three related GPCRs, mu, delta, and kappa, that activate Gi/o to reduce intracellular cAMP levels. Most clinically used opioids function by activation of the mu opioid receptor.
Growth Properties: Adherent
Host Cell: Chem-5
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of OPRM1.2. Protein expression: OPRM1 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by DAMGO: ~ 67nM.
Application: calcium flux assay, ligand binding assay
Sub-type: Opioid
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: 90% heat-inactivated FBS + 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: OPRM1 opioid receptor, mu 1 [ Homo sapiens ]
Official Symbol: OPRM1
Synonyms: OPRM1; opioid receptor, mu 1; mu-type opioid receptor; MOR1; MOP; hMOP; M-OR-1; mu opiate receptor; mu opioid receptor hMOR-1a; mu opioid receptor splice variant hMOR-1S; mu opioid receptor splice variant hMOR-1Z; mu opioid receptor splice variant hMOR-1b; mu opioid receptor splice variant hMOR-1Y2; mu opioid receptor splice variant hMOR-1Y3; MOR; LMOR; OPRM; KIAA0403;
Gene ID: 4988
mRNA Refseq: NM_000914
Protein Refseq: NP_000905
MIM: 600018
UniProt ID: P35372
Chromosome Location: 6q24-q25
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; G-protein activation, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class A Rhodopsin-like, organism-specific biosystem; IL4-mediated signaling events, organism-specific biosystem;
Function: G-protein alpha-subunit binding; G-protein coupled receptor activity; beta-endorphin receptor activity; morphine receptor activity; protein binding; 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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