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Human PTGER3-FLAG Stable Cell Line-CHO

Cat.No.: CSC-RG0365
Cell Line Description: CHO-HuPTGER3-FLAG is clonally-derived from a CHO-K1 cell line which has been transfected with a human prostaglandin E receptor 3 (PTGER3) tagged in the N-terminus with FLAG to allow stably express of the human PTGER3 tagged in the N-terminus with FLAG. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: Prostaglandin E2 (PGE2) is involved in a number of physiologic and pathophysiologic events in many tissues of the body. The biologic effects of PGE2 are mediated through interaction with specific membrane-bound G protein-coupled prostanoid EP receptors. EP3 receptor (or PTGER3) is expressed as multiple transcripts through alternative splicing, with each transcript showing a different tissue-specific distribution. PGE2 may mediate fever generation in response to both endogenous and exogenous pyrogens by acting at the EP3 receptor. EP3-mediated neuronal pathways converge at corticotropin-releasing hormone containing neurons in the paraventricular nucleus of the hypothalamus to induce HPA axis activation during sickness. At cellular level, EP3 has been shown to couple to both Gs and Gi/o types of the heterotrimeric G proteins to stimulate or inhibit intracellular cAMP synthesis. This cell line is responsive to pertussis toxin (PTX) treatment, indicating its coupling to both types of G proteins.
Growth Properties: Adherent
Morphology: Epithelial-like
Host Cell: CHO-K1
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human PTGER3.2. Protein expression: PTGER3 expression in this cell line has been validated by WB.3. Functional validation: Functional assays.
Sub-type: Prostanoid
Propagation: Complete growth medium: DMEM-F12, 10% FBS, 10 μg/mL puromycinAtmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Starting Cells From Frozen Cell Stock: 1. Remove the packaging cell lines from liquid nitrogen and carry out a quick thaw. Float the cells in the 37°C water bath for 2 minutes until nearly (80%) thawed. Once cells are thawed, it is important to dilute the cells 1:10 in growth media immediately to reduce the potentially toxic effects of the DMSO preservative on the cells.2. Clean the outside of the vial with 70% ethanol before opening.3. Remove the cells from the vial and add slowly into a 15ml conical tube containing 10 ml pre-warmed media. 4. Centrifuge for 3 minutes 1000 xg to pellet cells and remove the supernatant.5. Add 14 ml of media and transfer cells to a T25 flask or a 100 mm culture dish.6. Place the cells in the 37°C incubator with 5% CO2.7. Allow incubation for 3-4 days to reach confluence. The cells will re-attach to the surface over a period of several days in culture at 37°C.
Subculturing: 1. Remove and discard culture medium.2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.5. Add appropriate aliquots of the cell suspension to new culture vessels.Incubate cultures at 37°C.Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:8 is recommended Medium Renewal: 2 to 3 times per week
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Complete growth medium 90%; DMSO, 10%
Storage: Liquid nitrogen
Preservation: 1. Detach cells from culture dish according to the Sub-Culture Procedure.2. Resuspend cells at a density of 5 x 10^6 cells/mL in freeze medium.Note: A T-75 culture flask typically yields enough cells for preparing two frozen vials.3. Aliquot 1 mL cells into cryogenic vials.4. Place vials in a freezing container and store at –80 °C overnight.5. Transfer vials to liquid nitrogen for long term storage. If properly stored, cells should remain stable for years.
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: PTGER3 prostaglandin E receptor 3 (subtype EP3) [ Homo sapiens ]
Official Symbol: PTGER3
Synonyms: PTGER3; prostaglandin E receptor 3 (subtype EP3); prostaglandin E2 receptor EP3 subtype; EP3; prostanoid EP3 receptor; PGE receptor, EP3 subtype; PGE2 receptor EP3 subtype; prostaglandin receptor (PGE-2); prostaglandin E receotor EP3 subtype 3 isoform; EP3e; EP3-I; EP3-II; EP3-IV; PGE2-R; EP3-III; MGC27302; MGC141828; MGC141829;
Gene ID: 5733
mRNA Refseq: NM_001126044
Protein Refseq: NP_001119516
MIM: 176806
UniProt ID: P43115
Chromosome Location: 1p31.2
Pathway: Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; Eicosanoid ligand-binding receptors, organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem;
Function: G-protein coupled receptor activity; ligand-activated sequence-specific DNA binding RNA polymerase II transcription factor activity; prostaglandin E receptor activity; prostaglandin E 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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