Human AKT1 knockout HCT116 cell line
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human AKT1 knockout HCT116 cell line (AB286570)
Western blot : Anti-AKT1 antibody [BLR245L] (ab314110) 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, ab314110 was shown to bind specifically to AKT1. A band was observed at 55 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in AKT1 knockout cell line. To generate this image, wild-type and AKT1 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-AKT1 antibody [BLR245L] (<a href='/en-us/products/primary-antibodies/akt1-antibody-blr245l-ab314110'>ab314110</a>) at 1/1000 dilution
Lane 1:
Wild-type HCT 116 cell lysate at 20 µg
Lane 2:
AKT1 knockout HCT 116 cell lysate at 20 µg
Lane 3:
HeLa cell lysate at 20 µg
Lane 4:
A549 cell lysate at 20 µg
Lane 5:
Recombinant Human AKT1 protein <a href='/en-us/products/proteins-peptides/recombinant-human-akt1-protein-ab116412'>ab116412</a> cell lysate at 0.2 µg
Secondary
All lanes:
Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Observed band size: 55 kDa
false
- NGS
Supplier Data
Next Generation Sequencing - Human AKT1 knockout HCT116 cell line (AB286570)
64 bp deletion after Arg67
Complementary Products
Primary Antibodies
AB81283
Anti-AKT1 (phospho S473) antibody [EP2109Y]
RabMAb|Recombinant|Lab Essentials|20ul selling size
![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT1 (phospho S473) antibody [EP2109Y] (AB81283)](https://content.abcam.com/products/images/akt1-phospho-s473-antibody-ep2109y-ab81283--immunohistochemistry-formalin-pfa-fixed-paraffin-embedded-sections-img98892.gif)
- 4
- 10
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
- 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
McCoY5a + 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
AKT1 regulates a broad spectrum of cellular functions through signaling pathways. It is an important player in the phosphatidylinositol 3-kinase (PI3K) and AKT pathway forming part of a complex involved in promoting cell survival and growth. AKT1 interacts closely with other proteins such as mTOR influencing cellular metabolism and autophagy. The phosphorylation state of AKT1 is critical for its activity with modifications like p-AKT S473 impacting its interaction with cellular substrates.
Pathways
AKT1 participates in the PI3K/AKT/mTOR signaling cascade instrumental in cell proliferation and metabolism. AKT1 integrates signals from insulin and growth factors modulating pathways that control cell growth and glucose uptake. This pathway involves proteins like mTOR and phospho-AKT which coordinate cellular responses to external stimuli. AKT1’s phosphorylation at serine 473 and the involvement of AKT1 E17K a gain-of-function mutation influence these pathways significantly.
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
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