BBC3 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 10 bp deletion in exon 2 and 1 bp insertion in exon 2 and 22 bp deletion in exon 2.
Images
Key facts
- Cell type
- HeLa
- Species or organism
- Human
- Tissue
- Cervix
- Form
- Liquid
- Knockout validation
- Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, 10 bp deletion in exon 2 and 1 bp insertion in exon 2 and 22 bp deletion in exon 2
Alternative names
BBC3_HUMAN, Bcl-2-binding component 3, JFY-1, PUMA alpha, PUMA/JFY1, p53 up-regulated modulator of apoptosis
Recommended products
BBC3 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 10 bp deletion in exon 2 and 1 bp insertion in exon 2 and 22 bp deletion in exon 2.
Key facts
- Cell type
- HeLa
- Species or organism
- Human
- Tissue
- Cervix
- Form
- Liquid
- Knockout validation
- Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, 10 bp deletion in exon 2 and 1 bp insertion in exon 2 and 22 bp deletion in exon 2
- Antibiotic resistance
- Puromycin 1µg/mL
- Disease
- Adenocarcinoma
- Concentration
Properties
- Gene name
- BBC3
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Sanger Sequencing, Western blot
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 2 US: 2
- Adherent/suspension
- Adherent
- Gender
- Female
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.
- Culture medium
- DMEM (High Glucose) + 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, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and 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
PUMA also known as BBC3 (Bcl-2-binding component-3) is a pro-apoptotic protein that plays important mechanical roles in promoting apoptosis. It has a mass of approximately 23 kDa. PUMA is expressed widely in various tissues including the lung liver and heart. The protein works mechanically by interacting with anti-apoptotic Bcl-2 family members releasing pro-apoptotic factors like Bax and Bak which engage mitochondria to trigger cell death.
- Biological function summary
PUMA functions as a pivotal mediator of apoptosis in response to diverse stimuli such as DNA damage and oncogenic stress. As part of the Bcl-2 family PUMA does not typically form a complex with other proteins but functions directly by binding and neutralizing anti-apoptotic Bcl-2 family proteins. This action results in the release of caspase-activating factors from mitochondria essential for programmed cell death.
- Pathways
Several cellular pathways incorporate PUMA to regulate apoptosis. PUMA is notably included in the p53 pathway where p53 transcriptionally activates PUMA following cellular stress or DNA damage. The presence of related proteins like Bax and Bim in the mitochondrial apoptotic pathway links these signals to the mitochondrial death machinery facilitating apoptosis.
- Associated diseases and disorders
PUMA plays significant roles in cancer and neurodegenerative conditions. PUMA overexpression induces excessive cell death relevant in the pathology of neurodegenerative diseases. In cancer PUMA's interactions with proteins like p53 highlight its involvement in tumor suppression suggesting that dysregulation of PUMA expression can contribute to tumorigenesis when anti-apoptotic signals predominate potentially leading to chemoresistance.
Product promise
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4 product images
Western blot - Human BBC3 (PUMA) knockout HeLa cell line (ab261832)
Lanes 1 - 4: Merged signal (red and green). Green - anti-Puma antibody observed at 25 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
anti-Puma antibody was shown to react with Puma in wild-type HeLa cells in Western blot with loss of signal observed in BBC3 knockout cell line ab261832 (BBC3 knockout cell lysate Human BBC3 (PUMA) knockout HeLa cell lysate ab256848). Wild-type HeLa and BBC3 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with anti-Puma antibody and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Anti-Puma antibody at 1/1000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: BBC3 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human BBC3 (PUMA) knockout HeLa cell line (ab261832)
Lane 3: A549 cell lysate at 20 µg
Lane 4: PC-3 cell lysate at 20 µg
Performed under reducing conditions.
Sanger Sequencing - Human BBC3 (PUMA) knockout HeLa cell line (ab261832)
Allele-2: 10 bp deletion in exon 2.
Sanger Sequencing - Human BBC3 (PUMA) knockout HeLa cell line (ab261832)
Allele-1: 22 bp deletion in exon 2.
Sanger Sequencing - Human BBC3 (PUMA) knockout HeLa cell line (ab261832)
Allele-3: 1 bp insertion in exon 2.
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