Human SPHK1 knockout A549 cell line
- KO Validated
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human SPHK1 knockout A549 cell line (AB301254)
Western blot : Rabbit Monoclonal[EPR24511-37] to SPHK1 ab302714 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 55 kDa in Wild-type A549 cell lysates with no signal observed at this size in SPHK1 knockout A549 cell line (ab301254). To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 100pc LICOR 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.
All lanes:
Western blot - Anti-SPHK1 antibody [EPR24511-37] (<a href='/en-us/products/primary-antibodies/sphk1-antibody-epr24511-37-ab302714'>ab302714</a>) at 1/1000 dilution
Lane 1:
Wild-type A549 at 20 µg
Lane 2:
Western blot - Human SPHK1 knockout A549 cell line (ab301254) at 20 µg
Lane 3:
HeLa at 20 µg
Lane 4:
Raji at 20 µg
Secondary
All lanes:
Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Predicted band size: 85 kDa
Observed band size: 85 kDa
false
- NGS
Lab
Next Generation Sequencing - Human SPHK1 knockout A549 cell line (AB301254)
1 bp insertion and 1 bp insertion after Phe34
Complementary Products
Primary Antibodies
AB302714
Anti-SPHK1 antibody [EPR24511-37]
20ul selling size|RabMAb|Recombinant|KO Validated
![Western blot - Anti-SPHK1 antibody [EPR24511-37] (AB302714)](https://content.abcam.com/products/images/sphk1-antibody-epr24511-37-ab302714--western-blot-img446286.jpg)
- 0
- 0
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, 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.
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
Within the cell SPHK1 participates in regulating cell survival proliferation and inflammation. It is part of the sphingolipid metabolic pathway which is not a traditional protein complex but more of a metabolic sequence. SPHK1 converts sphingosine a lipid signaling molecule into S1P which acts as a ligand for G-protein-coupled receptors. Through this conversion SPHK1 influences diverse cellular functions.
Pathways
This protein actively participates in the sphingolipid signaling pathway and the apoptosis pathway. In sphingolipid signaling S1P acts alongside ceramide and other sphingolipids to modulate cell response. SPHK1's interplay with the ceramide-sphingosine-S1P rheostat can determine cell fate by tipping the balance towards survival or apoptosis. In the apoptosis pathway SPHK1 serves as a regulator of cell death with its function connected to proteins such as Bcl-2 and Bax which are primary apoptosis regulators.
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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