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Human SSR2/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0756
Cell Line Description: This human sst2-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant sst2 expression on the cell surface and contains optimal levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between sst2 and its ligands.
Background: Somatostatin (sst) is a multifunctional peptide with two biologically active forms, sst-14 and sst-28, which are synthesized in neurons throughout the brain as well as in peripheral tissues such as the pancreas and the gut. SST exerts a diverse array of effects that include inhibition of endocrine secretion, modulation of neurotransmission, and regulation of cell proliferation by stimulating a family of five G-protein-coupled receptors. Somatostatin receptor sst2 mRNA is predominantly expressed in central nervous system. Study using sst2 knock-out mice has found the increased anxiety-related behaviour while locomotor and exploratory activity was decreased in stress-inducing situations (coupled with an increase in pituitary ACTH release, a regulator of the stress response). In the periphery, inhibition of glucagon release by sst in mouse islets is primarily mediated via sst2. In addition, endogenous sst functions through sst2 to suppress gastric acid secretion through inhibition of gastrin activity.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of SSR2. 2. Protein expression: SSR2 in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by somatostatin: ~13.4nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Somatostatin
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: SSR2 signal sequence receptor, beta (translocon-associated protein beta) [ Homo sapiens ]
Official Symbol: SSR2
Synonyms: SSR2; signal sequence receptor, beta (translocon-associated protein beta); translocon-associated protein subunit beta; TLAP; TRAPB; SSR-beta; translocon-associated protein beta; signal sequence receptor subunit beta; HSD25; TRAP-BETA; DKFZp686F19123;
Gene ID: 6746
mRNA Refseq: NM_003145
Protein Refseq: NP_003136
MIM: 600867
UniProt ID: P43308
Chromosome Location: 1q21-q23
Pathway: Gene Expression, organism-specific biosystem; Metabolism of proteins, organism-specific biosystem; Protein processing in endoplasmic reticulum, organism-specific biosystem; Protein processing in endoplasmic reticulum, conserved biosystem; SRP-dependent cotranslational protein targeting to membrane, organism-specific biosystem; Translation, organism-specific biosystem; Translocon-associated protein (TRAP) complex, organism-specific biosystem;

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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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