TSC2 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided.
Images
Key facts
- Cell type
- A549
- Species or organism
- Human
- Tissue
- Lung
- Form
- Liquid
- Knockout validation
- Next Generation Sequencing, Western blot
Alternative names
FLJ43106, LAM, OTTHUMP00000158940, OTTHUMP00000198394, OTTHUMP00000198395, PPP1R160, Protein phosphatase 1, regulatory subunit 160, TSC complex subunit 2, TSC2_HUMAN, TSC4, TSC4 gene, formerly, TSC4, formerly, Tuberin, Tuberous sclerosis 2, Tuberous sclerosis 2 protein, Tuberous sclerosis 2 protein homolog
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TSC2 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided.
Key facts
- Cell type
- A549
- Species or organism
- Human
- Tissue
- Lung
- Form
- Liquid
- Knockout validation
- Next Generation Sequencing, Western blot
- Disease
- Carcinoma
- Concentration
Properties
- Gene name
- TSC2
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Next Generation Sequencing, Western blot
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 1 US: 1
- Adherent/suspension
- Adherent
- Gender
- Male
Handling procedures
- Initial handling guidelines
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.- Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not allow the cell density to exceed 7x104 cells/cm2.
- Culture medium
- F-12K + 10% FBS
- Cryopreservation medium
- Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- -196°C
- Appropriate long-term storage conditions
- -196°C
Notes
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
Tuberin also known as TSC2 is a tumor suppressor protein with a role in cell growth regulation. It has a molecular mass of approximately 200 kDa. This protein forms a critical component of the TSC1-TSC2 complex and is expressed widely in various tissues including the brain kidney heart and skin. Tuberin possesses GTPase-activating protein (GAP) activity targeting specifically the small GTPase protein Rheb which leads to inhibition of the mammalian target of rapamycin complex 1 (mTORC1).
- Biological function summary
Tuberin helps control cell size and proliferation. It does this by modulating the mTOR signaling pathway. This modulation is essential for maintaining cellular homeostasis and energy balance particularly under conditions of nutrient scarcity. Tuberin as part of the TSC1-TSC2 complex also influences cell cycle progression and protein synthesis. Its functions are vital in preventing abnormal cell growth and division which is particularly important in organs where rapid cell turnover occurs.
- Pathways
Tuberin plays a central role in the mTOR signaling pathway which affects cell growth autophagy and metabolism. It directly interacts with hamartin (TSC1) to form the TSC1-TSC2 complex which regulates Rheb activity. By inhibiting Rheb Tuberin helps control the mTORC1 pathway linking it to signals such as growth factors stress energy status and amino acid availability. This coordination is important for the proper balance between cell growth and catabolism.
- Associated diseases and disorders
Tuberin is mainly associated with Tuberous Sclerosis Complex (TSC) and certain forms of epilepsy. Mutations or loss of function in TSC2 can lead to benign tumor formations in multiple organs including the brain kidneys and skin. It is also implicated in the development of lymphangioleiomyomatosis (LAM) a rare lung disease. Tuberin's interaction with mTORC1 highlights its connection with these conditions as hyperactivation of the mTOR pathway contributes significantly to the pathogenesis.
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3 product images
Western blot - Human TSC2 (Tuberin) knockout A549 cell line (ab277867)
Western blot: Anti-TSC2 antibody [EP1107Y] (Anti-Tuberin antibody [EP1107Y] ab52936) staining at 1/50000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Tuberin antibody [EP1107Y] ab52936 was shown to bind specifically to TSC2. A band was observed at 200 kDa in wild-type A549 cell lysates with no signal observed at this size in TSC2 knockout cell line. To generate this image, wild-type and TSC2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-Tuberin antibody [EP1107Y] (Anti-Tuberin antibody [EP1107Y] ab52936) at 1/50000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: TSC2 knockout A549 cell lysate at 20 µg
Lane 3: Wild-type HAP1 cell lysate at 20 µg
Lane 4: TSC2 knockout HAP1 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Western blot - Human TSC2 (Tuberin) knockout A549 cell line (ab277867)
Western blot: Anti-TSC2 antibody [EPR22886-235] (Anti-Tuberin antibody [EPR22886-235] ab255612) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Tuberin antibody [EPR22886-235] ab255612 was shown to bind specifically to TSC2. A band was observed at 200 kDa in wild-type A549 cell lysates with no signal observed at this size in TSC2 knockout cell line. To generate this image, wild-type and TSC2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-Tuberin antibody [EPR22886-235] (Anti-Tuberin antibody [EPR22886-235] ab255612) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: TSC2 knockout A549 cell lysate at 20 µg
Lane 3: Wild-type HAP1 cell lysate at 20 µg
Lane 4: TSC2 knockout HAP1 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Next Generation Sequencing - Human TSC2 (Tuberin) knockout A549 cell line (ab277867)
56 bp deletion after Gln62 of the WT protein.
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