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Human CCKAR-SNAP Stable Cell Line-CHO

Cat.No.: CSC-RG0045
Cell Line Description: CHO-HuCCKAR-SNAP cell line is clonally-derived from a CHO cell line, which has been transfected with a Human Cholecystokinin CCK1 receptor (CCKAR) tagged in the N-terminus with SNAP to allow stably express of the human CCKAR tagged in the N-terminus with SNAP protein. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: This gene encodes a G-protein coupled receptor that binds non-sulfated members of the cholecystokinin (CCK) family of peptide hormones. This receptor is a major physiologic mediator of pancreatic enzyme secretion and smooth muscle contraction of the gallbladder and stomach. In the central and peripheral nervous system this receptor regulates satiety and the release of beta-endorphin and dopamine.
Growth Properties: Adherent
Morphology: Epithelial-like
Host Cell: CHO
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human CCKAR.2. Protein expression: CCKAR expression in this cell line has been validated by WB.3. Functional validation: Ligand binding assay; Receptor dimerization assays; IP-One assay; Internalization assay; Phospho-ERK assay
Sub-type: Cholecystokinin
Propagation: Complete growth medium: Ham"s F12, 10% dFBS, 50U/ml penicillin, 50μg/ml streptomycin, 2mM Hepes Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Starting Cells From Frozen Cell Stock: Thaw the vials of cryopreserved cells rapidly in a water bath at 37°C. Carefully transfer the cells to a cell culture vessel containing the appropriate volume of pre-warmed culture medium without G418 so that cell density reaches 4 x 10^4 to 8 x 10^4 cells/cm2. The day after, ensure that cells have properly attached and carefully replace with fresh culture medium supplemented with G418. Allow cells to grow until 80% confluency then transfer in a new cell culture vessel. Note that final DMSO concentration must not exceed 0.5%.
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.6. 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: CCKAR cholecystokinin A receptor [ Homo sapiens ]
Official Symbol: CCKAR
Synonyms: CCKAR; cholecystokinin A receptor; cholecystokinin receptor type A; CCK-AR; CCK-A receptor; cholecystokinin-1 receptor; cholecystokinin type-A receptor; CCK-A; CCKRA; CCK1-R;
Gene ID: 886
mRNA Refseq: NM_000730
Protein Refseq: NP_000721
MIM: 118444
UniProt ID: P32238
Chromosome Location: 4
Pathway: Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; 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; GPCRs, Class A Rhodopsin-like, organism-specific biosystem;
Function: G-protein coupled receptor activity; cholecystokinin 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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