RB1 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided. Knockout.
Images
Key facts
- Cell type
- HCT116
- Species or organism
- Human
- Tissue
- Colon
- Form
- Liquid
- Knockout validation
- Next Generation Sequencing, Western blot
- Mutation description
- Knockout.
Alternative names
Exon 17 tumor GOS561 substitution mutation causes premature stop, GOS563 exon 17 substitution mutation causes premature stop, OSRC, PP105, PPP1R130, PRB, Prepro retinoblastoma associated protein, Protein phosphatase 1 regulatory subunit 130, RB transcriptional corepressor 1, RB1, RB1 gene, RB_HUMAN, Retinoblastoma 1, Retinoblastoma suspectibility protein, Retinoblastoma-associated protein, p105-Rb, pp110
Recommended products
RB1 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided. Knockout.
Key facts
- Cell type
- HCT116
- Species or organism
- Human
- Tissue
- Colon
- Form
- Liquid
- Knockout validation
- Next Generation Sequencing, Western blot
- Mutation description
- Knockout.
- Disease
- Carcinoma
- Concentration
Properties
- Gene name
- RB1
- 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.
- Culture medium
- McCoY5a + 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
Retinoblastoma protein (Rb) also known as pRb is an important regulatory protein with a molecular weight of approximately 110 kDa. It is mainly expressed in the nucleus of cells. Rb functions as a tumor suppressor by controlling the cell cycle progression from G1 to S phase. Rb becomes active when it is dephosphorylated allowing it to bind and inhibit E2F transcription factors consequently preventing the transcription of genes essential for S phase entry.
- Biological function summary
Retinoblastoma protein influences cellular proliferation differentiation and apoptosis. It acts within a larger protein complex modulating various cellular responses. When functional Rb halts uncontrolled cell division important for maintaining normal tissue homeostasis. In its phosphorylated state known as phospho-Rb or phospho-Rb E182 it loses its regulatory capabilities which can lead to unrestrained cell cycle progression.
- Pathways
Several involve the retinoblastoma protein. One key pathway is the p53 pathway which Rb interacts with to influence cellular outcomes like cell-cycle arrest and apoptosis. Rb cooperates with proteins like p21 to implement these processes. Additionally it is involved in the cyclin D-dependent kinase 4 (CDK4) pathway which modulates its phosphorylation state influencing binding interactions and cell cycle control.
- Associated diseases and disorders
Dysregulation of retinoblastoma protein is commonly associated with cancer particularly retinoblastoma and breast cancer. In retinoblastoma mutations in the Rb gene directly lead to uncontrolled cell proliferation due to the absence of functional Rb protein. Additionally Rb's connection to the E2F family of transcription factors can become disrupted contributing to oncogenesis in other cancers.
Product promise
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3 product images
Western blot - Human RB1 knockout HCT116 cell line (ab286657)
Western blot: Anti-RB1 antibody (Anti-Rb antibody ab226979) staining at 1/1500 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-Rb antibody ab226979 was shown to bind specifically to RB1. A band was observed at 120 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in RB1 knockout cell line. To generate this image, wild-type and RB1 knockout HCT 116 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-Rb antibody (Anti-Rb antibody ab226979) at 1/1500 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lanes 1 - 4: Western blot - Human RB1 knockout HCT116 cell line (ab286657)
Lane 2: RB1 knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type A549 ab288558 cell lysate at 20 µg
Lane 4: RB1 knockout A549 Human RB1 knockout A549 cell line ab286470 cell lysate at 20 µg
SecondaryLanes 1 - 4: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 4: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 120 kDa
Next Generation Sequencing - Human RB1 knockout HCT116 cell line (ab286657)
8 bp deletion after Thr139 of the WT protein
Western blot - Human RB1 knockout HCT116 cell line (ab286657)
Western blot: Rabbit monoclonal [EPR17512] to Rb Anti-Rb antibody [EPR17512] ab181616 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 106 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in RB1 knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.
All lanes: Western blot - Anti-Rb antibody [EPR17512] (Anti-Rb antibody [EPR17512] ab181616) at 1/1000 dilution
Lane 1: Wild-type HCT 116 at 20 µg
Lane 2: Western blot - Human RB1 knockout HCT116 cell line (ab286657) at 20 µg
Lane 3: Wild-type A549 ab288558 at 20 µg
Lane 4: Western blot - Human RB1 knockout A549 cell line (Human RB1 knockout A549 cell line ab286470) at 20 µg
Lane 5: HEK-293 at 20 µg
SecondaryAll lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 106 kDa
Observed band size: 106 kDa
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