Human CRBN knockout MCF7 cell line
- Advanced Validation
- What is this?
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- CRBN KO validation: Next Generation Sequencing (NGS),Western blot
- Concentration: 1 million cells/vial
- WB
Lab
Western blot - Human CRBN knockout MCF7 cell line (AB289426)
Western blot : Anti-CRBN antibody [4D6] (ab244223) staining at 0.2 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab244223 was shown to bind specifically to CRBN. A band was observed at 51 kDa in wild-type MCF7 cell lysates with no signal observed at this size in CRBN knockout cell line. To generate this image, wild-type and CRBN knockout MCF7 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
All lanes:
Western blot - Anti-CRBN antibody [4D6] (<a href='/en-us/products/primary-antibodies/crbn-antibody-4d6-ab244223'>ab244223</a>) at 0.2 µg/mL
Lane 1:
Wild-type MCF7 cell lysate at 20 µg
Lane 2:
CRBN knockout MCF7 cell lysate at 20 µg
Lane 3:
Wild-type A549 ab288558 cell lysate at 20 µg
Lane 4:
CRBN knockout A549 ab288959 cell lysate at 20 µg
Secondary
All lanes:
Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution
Observed band size: 51 kDa
false
- NGS
Supplier Data
Next Generation Sequencing - Human CRBN knockout MCF7 cell line (AB289426)
83 bp deletion after Ser76 of the WT protein.
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Reactivity data
Product details
Although we aim to provide customers with a homozygous clone, feasibility will be dependent on the biology of the protein. Should only heterozygous edits be achieved, you will be notified of the outcome and be asked to confirm whether the cell line is acceptable. All clones will be accompanied with DNA sequencing data, and the mutation description.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
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.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
Cereblon plays a role in the regulation of protein stability and degradation. It forms part of the CRL4-CRBN complex which tags specific proteins for ubiquitination and subsequent proteasomal degradation. This function is critical for maintaining cellular protein balance. Additionally CRBN modulates immune response and has immunomodulator properties influencing cell-mediated responses.
Pathways
CRBN participates in the ubiquitin-proteasome pathway which regulates protein turnover and quality control within cells. Moreover CRBN is involved in immune signaling pathways partly by modulating the expression of various cytokines and immune-related proteins. It interacts with proteins such as DDB1 and CUL4 within these pathways impacting cellular responses to stress and immune challenges.
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Female
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com