Human KEAP1 knockout A549 cell line
- Advanced Validation
- What is this?
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KEAP1 KO cell line available to order. KO validated by Next Generation Sequencing. Free of charge wild type control provided.
View Alternative Names
Cytosolic inhibitor of Nrf2, INrf 2, KEAP1_HUMAN, KIAA0132, KLHL 19, Kelch like family member 19, Kelch-like ECH-associated protein 1, Kelch-like protein 19, MGC10630, MGC1114, MGC20887, MGC4407, MGC9454
- WB
Lab
Western blot - Human KEAP1 knockout A549 cell line (AB286671)
Western blot : Anti-KEAP1 antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, anti-KEAP1 antibody was shown to bind specifically to KEAP1. A band was observed at 70 kDa in wild-type A549 cell lysates with no signal observed at this size in KEAP1 knockout cell line. To generate this image, wild-type and KEAP1 knockout A549 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:
Anti-KEAP1 antibody at 1/1000 dilution
Lane 1:
Wild-type A549 cell lysate at 20 µg
Lane 2:
Western blot - Human KEAP1 knockout A549 cell line (ab286671)
Lane 2:
KEAP1 knockout A549 cell lysate at 20 µg
Lane 3:
Wild-type HCT 116 cell lysate at 20 µg
Lane 4:
Western blot - Human KEAP1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-keap1-knockout-hct116-cell-line-ab286484'>ab286484</a>)
Lane 4:
KEAP1 knockout HCT 116 <a href='/en-us/products/cell-lines/human-keap1-knockout-hct116-cell-line-ab286484'>ab286484</a> cell lysate at 20 µg
Secondary
Lanes 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
Observed band size: 70 kDa
false
- NGS
Lab
Next Generation Sequencing - Human KEAP1 knockout A549 cell line (AB286671)
125 bp deletion after Asp 86 of WT protein
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.
Recommended control: Human wild-type A549 cell line (ab288558). 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.
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
- 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.
- Do not allow the cell density to exceed 7x104 cells/cm2.
Culture medium
F-12K + 10% FBS
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
Keap1 serves as a sensor for oxidative stress within the cell. It constitutes part of a larger biological complex that includes the NRF2 protein and the cullin-3-based E3 ligase complex. In the presence of oxidative or electrophilic stress modifications in Keap1 cysteine thiol groups lead to conformational changes inhibiting Keap1’s ability to target NRF2 for degradation. This results in the accumulation and activation of NRF2 which migrates to the nucleus where it promotes the expression of antioxidant response element (ARE)-driven genes important for cellular defense mechanisms.
Pathways
Keap1 plays a central role in the NRF2-ARE signaling pathway a major player in the antioxidant response of cells. This pathway connects to various processes including detoxification and the regulation of oxidative stress. Keap1 interacts with other proteins such as cullin-3 which is vital for ubiquitin tagging of NRF2. Additionally Keap1 influences the WNT signaling pathway through its interactions with other proteins impacting processes related to cellular proliferation and differentiation.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
Target data
Complementary Products
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Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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