CDKN1A KO cell line available now. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control available.
Images
Key facts
- Cell type
MCF7
- Species or organism
Human
- Tissue
Breast
- Form
Liquid
- Knockout validation
Next Generation Sequencing, Sanger Sequencing, Western blot
Alternative names
CAP20, CDK-interacting protein 1, CDKI, CDKN1, CDKN1A, CDN1A_HUMAN, Cyclin Dependent Kinase Inhibitor 1A, Cyclin-dependent kinase inhibitor 1, Cyclin-dependent kinase inhibitor 1A (P21), Cyclin-dependent kinase inhibitor 1A (p21, Cip1), DNA Synthesis Inhibitor, MDA-6, Melanoma differentiation-associated protein, Melanoma differentiation-associated protein 6, P21 protein, PIC 1, SDI1, SLC12A9, WAF1, Wild type p53 activated fragment 1 (WAF1), Wildtype p53-activated fragment 1, p21CIP1, p21Cip1/Waf1, p21WAF
Recommended products
CDKN1A KO cell line available now. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control available.
Alternative names
CAP20, CDK-interacting protein 1, CDKI, CDKN1, CDKN1A, CDN1A_HUMAN, Cyclin Dependent Kinase Inhibitor 1A, Cyclin-dependent kinase inhibitor 1, Cyclin-dependent kinase inhibitor 1A (P21), Cyclin-dependent kinase inhibitor 1A (p21, Cip1), DNA Synthesis Inhibitor, MDA-6, Melanoma differentiation-associated protein, Melanoma differentiation-associated protein 6, P21 protein, PIC 1, SDI1, SLC12A9, WAF1, Wild type p53 activated fragment 1 (WAF1), Wildtype p53-activated fragment 1, p21CIP1, p21Cip1/Waf1, p21WAF
Key facts
- Cell type
MCF7
- Species or organism
Human
- Tissue
Breast
- Form
Liquid
- Knockout validation
Next Generation Sequencing, Sanger Sequencing, Western blot
- Disease
Adenocarcinoma
- Concentration
Properties
- Gene name
CDKN1A
- Gene editing type
Knockout
- Gene editing method
CRISPR technology
- Knockout validation
Next Generation Sequencing, Sanger Sequencing, Western blot
Cell culture
- Biosafety level
EU: 1 US: 1
- 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
- Slow to trypsinise.
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 5-7x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
- Culture medium
MEM + 10% FBS + 0.01 mg/ml bovine insulin
- 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 long-term storage conditions
-196°C
Notes
Recommended control: Human wild-type MCF7 cell line (Human wild-type MCF7 cell line ab288560). 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: MEM + 10% FBS + 0.01 mg/mL bovine insulin
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 5-7x104 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 5-7x104 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
- Activity summary
The p21 protein also known as CDKN1A is an important regulator of cell cycle progression. It acts as a cyclin-dependent kinase inhibitor where it binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes. The molecular weight of p21 is approximately 21 kDa. Cellular expression of p21 is diverse; it occurs in various tissues reflecting its broad role in maintaining cell cycle control. This protein's expression level is often assessed using techniques such as p21 western blotting p21 ELISA and p21 IHC staining.
- Biological function summary
P21 plays a critical role in cell cycle regulation and DNA damage response. It associates with the p53 tumor suppressor protein forming an essential part of the p53 signaling pathway under stress conditions. p21 arrests cells in G1 phase allowing DNA repair or leading to cellular senescence. Through these mechanisms p21 integrates signals from stress and damage emphasizing its importance as a mediator in cellular checkpoint control.
- Pathways
P21 is closely involved in the p53 and the Transforming Growth Factor-beta (TGF-β) signaling pathways. This integration helps in transmitting growth-inhibitory signals. p21 interacts with proteins such as p53 and cyclins ensuring precise regulation of the cell cycle and growth arrest when necessary. Triggered by p53 activation p21 contributes to preventing undisturbed cell proliferation acting as a safeguard against tumor formation.
- Associated diseases and disorders
P21's deregulation associates with cancer and age-related diseases. Its impairment is a common feature in many cancers often linked with defects in the p53 pathway. Reduced p21 expression can contribute to unchecked cell division and tumor progression. Moreover in age-related diseases p21's role in promoting cellular senescence connects it with degenerative conditions. The intricate relationship between p21 and proteins such as p53 and cyclins highlights its potential as a therapeutic target for these disorders.
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4 product images
Next Generation Sequencing - Human CDKN1A knockout MCF7 cell line (ab288200)
221 bp deletion after Gly123 of the WT protein
Western blot - Human CDKN1A knockout MCF7 cell line (ab288200)
Western blot: Anti-CDKN1A antibody [CIP1/823] (Anti-p21 antibody [CIP1/823] ab220206) staining at 1 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-p21 antibody [CIP1/823] ab220206 was shown to bind specifically to CDKN1A. A band was observed at 21 kDa in wild-type cell lysates with no signal observed at this size in CDKN1A knockout cell lines. To generate this image, wild-type and CDKN1A knockout cell lysates were analysed. CDKN1A knockout cell lines in HCT 116 (Human CDKN1A knockout HCT116 cell line ab288187), MCF7 (ab288200) and A549 (Human CDKN1A knockout A549 cell line ab288213) backgrounds were used. 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-p21 antibody [CIP1/823] (Anti-p21 antibody [CIP1/823] ab220206) at 1 µg/mL
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: CDKN1A knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type MCF7 cell lysate at 20 µg
Lane 4: CDKN1A knockout MCF7 cell lysate at 20 µg
Lane 5: Wild-type A549 cell lysate at 20 µg
Lane 6: CDKN1A knockout A549 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 18 kDa
Observed band size: 21 kDa
Western blot - Human CDKN1A knockout MCF7 cell line (ab288200)
Western blot: Anti-CDKN1A antibody [EPR362] (Anti-p21 antibody [EPR362] ab109520) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-p21 antibody [EPR362] ab109520 was shown to bind specifically to CDKN1A. A band was observed at 21 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CDKN1A knockout cell line. To generate this image, wild-type and CDKN1A 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-p21 antibody [EPR362] (Anti-p21 antibody [EPR362] ab109520) at 1/1000 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: CDKN1A knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type MCF7 cell lysate at 20 µg
Lane 4: CDKN1A knockout MCF7 cell lysate at 20 µg
Lane 5: Wild-type A549 cell lysate at 20 µg
Lane 6: CDKN1A knockout A549 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.
Predicted band size: 21 kDa
Observed band size: 21 kDa
Sanger Sequencing - Human CDKN1A knockout MCF7 cell line (ab288200)
221bp deletion after Ala 5 of the WT protein.
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