Human A-Raf knockout HCT116 cell line
- Advanced Validation
- What is this?
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(0 Publication)
- WB
Supplier Data
Western blot - Human A-Raf knockout HCT116 cell line (AB286752)
Anti-A-Raf antibody [EPR16208] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab200653 was shown to bind specifically to A-Raf. A band was observed at 67 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in A-Raf knockout cell line ab286752. To generate this image, wild-type and A-Raf 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-A-Raf antibody [EPR16208] (<a href='/en-us/products/primary-antibodies/a-raf-antibody-epr16208-ab200653'>ab200653</a>) at 1/1000 dilution
Lane 1:
Wild-type HCT 116 cell lysate at 20 µg
Lane 2:
A-Raf knockout HCT 116 cell lysate at 20 µg
Lane 3:
HEK-293 cell lysate at 20 µg
Lane 4:
Raji cell lysate at 20 µg
Secondary
All lanes:
Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Predicted band size: 68 kDa
Observed band size: 67 kDa
false
- NGS
Lab
Next Generation Sequencing - Human A-Raf knockout HCT116 cell line (AB286752)
131 bp deletion after the Lys1223 of the WT protein
Complementary Products
![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-A-Raf antibody [EPR16208] (AB200653)](https://content.abcam.com/products/images/a-raf-antibody-epr16208-ab200653--immunohistochemistry-formalin-pfa-fixed-paraffin-embedded-sections-img107369.jpg)
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 HCT116 cell line (ab288559). 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.
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
The A-Raf protein participates in the regulation of cell growth and survival. It forms part of the MAPK/ERK signaling cascade which is essential for transmitting mitogenic signals from the cell surface to the nucleus. A-Raf acts as a modulator within this complex impacting the overall signaling output. The protein is intimately involved in the regulation of the developmental processes influencing cell fate and function.
Pathways
The A-Raf protein is central to the MAPK/ERK pathway a critical signaling pathway involved in the regulation of cellular responses to growth signals. This pathway includes key components like the proteins B-Raf and C-Raf with A-Raf playing specific roles in the modulation and fine-tuning of this signal transmission. It helps mediate signals that control cell division differentiation and secretion ensuring appropriate cellular responses to external stimuli.
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'.
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