APAF1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided.
Images
Key facts
- Cell type
- HCT116
- Species or organism
- Human
- Tissue
- Colon
- Form
- Liquid
- Knockout validation
- Western blot
Alternative names
APAF_HUMAN, Apoptotic peptidase activating factor 1, Apoptotic protease activating factor, Apoptotic protease-activating factor 1, CED 4, KIAA0413
Recommended products
APAF1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided.
Key facts
- Cell type
- HCT116
- Species or organism
- Human
- Tissue
- Colon
- Form
- Liquid
- Knockout validation
- Western blot
- Disease
- Carcinoma
- Concentration
Properties
- Gene name
- APAF1
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Western blot
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.
- 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
Recommended control: Human wild-type HCT116 cell line (ab273730). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: McCoY5a + 10% FBS
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. Based on cell count, seed cells in an appropriate cell culture 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.
4. Incubate the culture at 37°C incubator with 5% CO2. 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.
• A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
• Cells should be passaged when they have achieved 80-90% confluence.
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
APAF1 also known as apoptotic protease activating factor 1 plays a mechanical role in apoptosis by forming a complex with other proteins to trigger cell death. The APAF1 protein has a molecular weight of approximately 130 kDa. It is expressed in many tissues but occurs highly in the brain and heart. APAF1 interacts with cytochrome c and procaspase-9 to promote the activation of the initiator caspase cascade.
- Biological function summary
APAF1 functions as a central component of the apoptosome a multi-protein complex important for programmed cell death. This process is essential for normal development and cellular homeostasis. In the apoptosome APAF1 recruits and activates procaspase-9 leading to the activation of caspase-3. This cascade assures that cells die in a controlled manner preventing damage to surrounding tissues.
- Pathways
APAF1 serves an important function in the intrinsic pathway of apoptosis. It mediates cytochrome c release from mitochondria a step essential for apoptosis initiation. Another important pathway involving APAF1 is the apoptotic signaling pathway. Proteins such as Bcl-2 family members and p53 influence APAF1's role in these pathways by regulating cytochrome c release and apoptosome assembly.
- Associated diseases and disorders
APAF1 is notably associated with neurodegenerative diseases and certain cancers. Dysregulation of APAF1 can lead to undesirable apoptosis contributing to neurodegenerative conditions like Parkinson’s disease. Conversely reduced APAF1 activity can result in tumor cell survival impacting cancer progression. Altered expression of APAF1 affects its interaction with proteins like Bcl-2 which can inhibit apoptosis and promote cancer cell growth.
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3 product images
Western blot - Human APAF1 knockout HCT116 cell line (ab300842)
Western blot: Anti-APAF1 antibody [EPR21112-102] (Anti-APAF1 antibody [EPR21112-102] ab234436) 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-APAF1 antibody [EPR21112-102] ab234436 was shown to bind specifically to APAF1. A band was observed at 140 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in APAF1 knockout cell line. To generate this image, wild-type and APAF1 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-APAF1 antibody [EPR21112-102] (Anti-APAF1 antibody [EPR21112-102] ab234436) at 1/1000 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: APAF1 knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type HAP1 cell lysate at 20 µg
Lane 4: APAF1 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 APAF1 knockout HCT116 cell line (ab300842)
Western blot: Anti-APAF1 antibody (Anti-APAF1 antibody ab2001) 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-APAF1 antibody ab2001 was shown to bind specifically to APAF1. A band was observed at 140 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in APAF1 knockout cell line. To generate this image, wild-type and APAF1 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-APAF1 antibody (Anti-APAF1 antibody ab2001) at 1/1500 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: APAF1 knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type HAP1 cell lysate at 20 µg
Lane 4: APAF1 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 APAF1 knockout HCT116 cell line (ab300842)
9 bp insertion after Asp65 of the WT protein
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