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Human CACNA1C/NEUROD1/CACNA2D1/KCNJ2 Stable Cell Line-HEK293

Cat.No.: CSC-RI0066
Cell Line Description: HEK293-HuCACNA1C/NEUROD1/CACNA2D1/KCNJ2 cell line is a hypotriploid human cell line, which has been transfected with a human calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C), a human neuronal differentiation 1 (NEUROD1), a human calcium channel, voltage-dependent, alpha 2/delta subunit 1 (CACNA2D1) and a human potassium inwardly-rectifying channel, subfamily J, member 2 (KCNJ2) to allow stably express of the human CACNA1C, NEUROD1, CACNA2D1 and KCNJ2. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: The human CACNA1C gene encodes the pore-forming subunits of Cav1.2. Inclusion of auxiliary subunits modulate gating and pharmacological properties, while the inwardly rectifying potassium channel allows the membrane potential to be altered with changes in extracellular K+. Cav1.2 channels expressed in heart, smooth muscle, neurons, and endocrine tissue are therapeutic targets in cardiac arrhythmia and hypertension.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific over-expression of human CACNA1C/NEUROD1/CACNA2D1/KCNJ2.2. Protein expression: CACNA1C/NEUROD1/CACNA2D1/KCNJ2 expression in this cell line has been validated by WB.3. Functional validation: FLIPR
Propagation: Complete growth medium: DMEM/F12/10% FBSAtmosphere: 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 1:6 to 1:10 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: NEUROD1 neuronal differentiation 1 [ Homo sapiens ]
Official Symbol: NEUROD1
Synonyms: NEUROD1; neuronal differentiation 1; NEUROD, neurogenic differentiation 1; neurogenic differentiation factor 1; beta cell E box transactivator 2; BETA2; BHF 1; bHLHa3; MODY6; NeuroD; neurogenic helix loop helix protein NEUROD; beta-cell E-box transactivator 2; class A basic helix-loop-helix protein 3; neurogenic helix-loop-helix protein NEUROD; basic helix-loop-helix transcription factor; BHF-1; NEUROD;
Gene ID: 4760
mRNA Refseq: NM_002500
Protein Refseq: NP_002491
MIM: 601724
UniProt ID: Q13562
Chromosome Location: 2q32
Pathway: Developmental Biology, organism-specific biosystem; Maturity onset diabetes of the young, organism-specific biosystem; Maturity onset diabetes of the young, conserved biosystem; Regulation of beta-cell development, organism-specific biosystem; Regulation of gene expression in beta cells, organism-specific biosystem; Regulation of gene expression in endocrine-committed (NEUROG3+) progenitor cells, organism-specific biosystem;
Function: E-box binding; RNA polymerase II activating transcription factor binding; RNA polymerase II transcription coactivator activity; chromatin binding; double-stranded DNA binding; protein binding; protein heterodimerization activity; protein heterodimerization activity; contributes_to sequence-specific DNA binding; sequence-specific DNA binding transcription factor activity; transcription coactivator activity; transcription factor binding;

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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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