CDK4 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 139 bp insertion in exon 2 and 2 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, 139 bp insertion in exon 2 and 2 bp deletion in exon 2
Alternative names
CDK4 protein, CDK4_HUMAN, CMM 3, Cell division kinase 4, Cell division protein kinase 4, Crk3, Cyclin-dependent kinase 4, MGC14458, Melanoma cutaneous malignant 3, PSK-J3, p34 cdk4
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CDK4 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 139 bp insertion in exon 2 and 2 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, 139 bp insertion in exon 2 and 2 bp deletion in exon 2
- Disease
- Adenocarcinoma
- Concentration
Properties
- Gene name
- CDK4
- 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
Cdk4 also known as Cyclin-dependent kinase 4 is a serine/threonine kinase involved in cell cycle regulation. Cdk4 has a molecular weight of approximately 34 kDa. This protein mainly locates in the nucleus of the cell and its expression occurs in diverse tissues. Cdk4 partners with D-type cyclins to exert its function which is essential for the transition from the G1 phase to the S phase of the cell cycle. Researchers often study Cdk4 using techniques like immunohistochemistry (IHC) to determine its expression and localization in different tissues.
- Biological function summary
Cdk4 plays a fundamental role in cell cycle regulation by forming a complex with D-type cyclins. Together they phosphorylate the retinoblastoma protein (Rb) resulting in the release of E2F transcription factors which promote the expression of genes necessary for DNA replication. The Cdk4/cyclin D complex regulates the cell's commitment to enter S phase and continue cell division. This regulation is key for normal cell proliferation and tissue homeostasis. Cdk4 activity is strictly controlled by INK4 family members acting as inhibitors and adding an extra regulation layer.
- Pathways
Cdk4 is an integral part of important signaling pathways like the cell cycle and PI3K/AKT pathways. In the cell cycle pathway Cdk4 relays signals downstream that drive the transition from the G1 to S phase through its interaction with cyclin D and Rb. In the PI3K/AKT pathway signaling can influence cyclin D expression indirectly affecting Cdk4 activity. Proteins such as Cdk6 closely relate to Cdk4 and often compensate or partner with Cdk4 in these pathways to maintain proper cell cycle progression.
- Associated diseases and disorders
Abnormalities in Cdk4 function or regulation relate to cancer and certain metabolic syndromes. Hyperactivity of Cdk4 due to overexpression or mutation frequently occurs in several cancers including melanoma and breast cancer. In these malignancies Cdk4 aberrantly drives excessive cell proliferation. Mutations or alterations in proteins such as p16 an inhibitor of Cdk4 are often found in these cancers highlighting the critical role of Cdk4 in disease progression. Understanding the role of Cdk4 allows researchers to develop targeted therapies such as Cdk4/6 inhibitors offering new treatment options.
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4 product images
Western blot - Human CDK4 knockout HeLa cell line (ab255378)
Lanes 1- 2: Merged signal (red and green). Green - Anti-Cdk4 antibody [EPR4513-32-7] ab108357 observed at 34 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.
Anti-Cdk4 antibody [EPR4513-32-7] ab108357 was shown to react with CDK4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255378 (knockout cell lysate Human CDK4 knockout HeLa cell lysate ab263780) was used. Wild-type HeLa and CDK4 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-Cdk4 antibody [EPR4513-32-7] ab108357 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (Anti-Cdk4 antibody [EPR4513-32-7] ab108357) at 1/1000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: CDK4 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human CDK4 knockout HeLa cell line (ab255378)
Performed under reducing conditions.
Predicted band size: 34 kDa, 55 kDa
Observed band size: 34 kDa, 55 kDa
Western blot - Human CDK4 knockout HeLa cell line (ab255378)
Lanes 1- 2: Merged signal (red and green). Green - Anti-Cdk4 antibody [EPR17525] ab199728 observed at 34 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.
Anti-Cdk4 antibody [EPR17525] ab199728 was shown to react with CDK4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255378 (knockout cell lysate Human CDK4 knockout HeLa cell lysate ab263780) was used. Wild-type HeLa and CDK4 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-Cdk4 antibody [EPR17525] ab199728 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-Cdk4 antibody [EPR17525] (Anti-Cdk4 antibody [EPR17525] ab199728) at 1/2000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: CDK4 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human CDK4 knockout HeLa cell line (ab255378)
Performed under reducing conditions.
Predicted band size: 34 kDa
Observed band size: 34 kDa
Sanger Sequencing - Human CDK4 knockout HeLa cell line (ab255378)
Allele-2: 139 bp insertion in exon 2.
Sanger Sequencing - Human CDK4 knockout HeLa cell line (ab255378)
Allele-1: 2 bp deletion in exon 2.
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