Human APTX (Aprataxin) knockout HeLa cell line
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- WB
Lab
Western blot - Human APTX (Aprataxin) knockout HeLa cell line (AB265118)
False colour image of Western blot : Anti-Aprataxin antibody staining at 1/500 dilution shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution shown in red. In Western blot ab154413 was shown to bind specifically to Aprataxin. A band was observed at 41 kDa in wild-type HeLa cell lysates with no signal observed at this size in APTX CRISPR-Cas9 edited cell line ab265118 (CRISPR-Cas9 edited cell lysate ab257837). The band observed in the CRISPR-Cas9 edited lysate lane below 41 kDa is likely to represent a truncated form of Aprataxin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image wild-type and APTX CRISPR-Cas9 edited HeLa cell lysates were analysed. First samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °. 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
Lanes 1, 2 and 4:
Western blot - Anti-Aprataxin antibody (<a href='/en-us/products/primary-antibodies/aprataxin-antibody-ab154413'>ab154413</a>) at 1/500 dilution
Lane 3:
Western blot - Anti-Aprataxin antibody (<a href='/en-us/products/primary-antibodies/aprataxin-antibody-ab154413'>ab154413</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
APTX CRISPR-Cas9 edited HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human APTX (Aprataxin) knockout HeLa cell line (ab265118)
Lane 3:
HepG2 cell lysate at 20 µg
Lane 4:
Saos-2 cell lysate at 20 µg
Predicted band size: 41 kDa
Observed band size: 41 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human APTX (Aprataxin) knockout HeLa cell line (AB265118)
Homozygous : 321 bp insertion in exon 1.
Complementary Products
Product details
We will provide viable cells that proliferate on revival.
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
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
DMEM (High Glucose) + 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
Aprataxin plays an important role in the DNA single-strand break repair processes. It functions within the DNA repair machinery often as part of complexes with other repair proteins. This protein mainly interacts with other partners like XRCC1 and PARP1 to facilitate accurate DNA repair. Its interaction ensures proper response to DNA damage safeguarding the genome's stability and function.
Pathways
Aprataxin is integrally connected to the base excision repair (BER) pathway and the DNA damage response pathway. These pathways are fundamental mechanisms for repairing damaged DNA and preserving cell viability. Within these pathways Aprataxin closely collaborates with related proteins like XRCC1 and PARP1. These associations highlight Aprataxin's role in ensuring the efficiency and success of DNA repair processes.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
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