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Human GABBR1/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0660
Cell Line Description: This human GABAB1b/GABAB2-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant GABAB1b/GABAB2 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 GABAB and its ligands.
Background: The neurotransmitter-aminobutyric acid (GABA) exerts its effects through an ion channel, GABAA, and a GPCR, GABAB. Functional GABAB is a heterodimer composed of the GABAB1 and GABAB2 subunits, which share 35% sequence identity and belong to the class 3 family of GPCRs. The GABAB1 subunit, which exists as splice variants GABAB1a and GABAB1b, binds directly to GABA and is required for agonist activation. The GABAB2 and GABAB1 subunits associate by formation of a coiled coil by their C-terminal tails; this association masks an ER retention sequence in GABAB1 to permit export from the ER and trafficking to the cell surface. In addition to its chaperone function, GABAB2 is the component that couples to Gi to reduce intracellular cAMP. Agonists of GABAB, such as baclofen, are used clinically for treatment of muscle spasticity, migraine headache and musculoskeletal pain.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of GABBR1.2. Protein expression: GABBR1 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by GABA: ~ 280 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: GABAB
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: GABBR1 gamma-aminobutyric acid (GABA) B receptor, 1 [ Homo sapiens ]
Official Symbol: GABBR1
Synonyms: GABBR1; gamma-aminobutyric acid (GABA) B receptor, 1; gamma-aminobutyric acid type B receptor subunit 1; GABA B receptor; GPRC3A; hGB1a; GABA-BR1; GABAB, subunit 1c; seven transmembrane helix receptor; GB1; GABABR1; GABBR1-3; dJ271M21.1.1; dJ271M21.1.2; FLJ92613;
Gene ID: 2550
mRNA Refseq: NM_001470
Protein Refseq: NP_001461
MIM: 603540
UniProt ID: Q9UBS5
Chromosome Location: 6p21.3
Pathway: Activation of G protein gated Potassium channels, organism-specific biosystem; Activation of GABAB receptors, organism-specific biosystem; Class C/3 (Metabotropic glutamate/pheromone receptors), organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; G protein gated Potassium channels, organism-specific biosystem; GABA B receptor activation, organism-specific biosystem; GABA receptor activation, organism-specific biosystem;
Function: G-protein coupled GABA receptor activity; protein binding; receptor activity; signal transducer activity; transcription factor binding;

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