ATG12 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided.
Images
Key facts
- Cell type
THP-1
- Species or organism
Human
- Tissue
Blood
- Form
Liquid
- Knockout validation
Western blot
Alternative names
APG12-like, APG12L, ATG12 autophagy related 12 homolog, ATG12 autophagy related 12 homolog (S. cerevisiae), ATG12_HUMAN, Apg12, Autophagy 12, Autophagy-related protein 12, FBR93, HAPG12, Ubiquitin-like protein ATG12
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ATG12 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided.
Key facts
- Cell type
THP-1
- Species or organism
Human
- Tissue
Blood
- Form
Liquid
- Knockout validation
Western blot
- Disease
Acute Monocytic Leukemia
- Concentration
Properties
- Gene name
ATG12
- Gene editing type
Knockout
- Gene editing method
CRISPR technology
- Knockout validation
Western blot
Quality control
- STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
EU: 1 US: 1
- Adherent/suspension
Suspension
- Gender
Male
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. Based on cell count, seed cells in an appropriate cell culture flask at a density of 4x105 cells/mL. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.
5. THP-1 cells recover slowly from cryopreservation and therefore may not be ready for subculture for a number of days. Cells should be left as much as possible over this time and only subcultured when the cell density reaches 8x105 cells/mL.Small amounts of fresh media can be added until cell number/viability improves.- Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- Cells should be seeded at 2x105 - 3x105 cells/mL and subcultured when they have reached 8x105 cells/mL.
- It is not recommended to allow the cell density to exceed 1x106 cells/mL.
- Culture medium
RPMI + 10% FBS + 0.05 mM beta-mercaptoethanol
- Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Storage
- Shipped at conditions
Dry Ice
- Appropriate short-term storage conditions
-196°C
- Appropriate long-term storage conditions
-196°C
Notes
Recommended control: Human wild-type THP-1 cell line (Human wild-type THP-1 cell line ab281894). 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.
Supplementary info
This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
ATG12 also known as Autophagy-related protein 12 plays a critical role in autophagy an essential cellular process for degrading and recycling cellular components. The molecular weight of ATG12 is around 14 kDa. It is part of the ATG12-conjugation system and is invariably expressed in various tissues with notable presence in the liver brain and heart. ATG12 forms a covalent bond with ATG5 in a ubiquitin-like conjugation pathway which is significant for this mechanism.
- Biological function summary
ATG12 is vital for the formation of autophagosomes which are double-membraned vesicles that sequester cellular components for degradation. It functions as part of a complex by binding to ATG5 and further associates with ATG16L1 to form a larger ATG12-ATG5-ATG16 complex. This complex localizes to the outer membrane of the growing autophagosome facilitating elongation and closure of the vesicle making it effective in the regulation of autophagy.
- Pathways
ATG12's interaction with other proteins like ATG5 places it in the macroautophagy pathway an important process maintaining cellular homeostasis and turnover of cellular components. In addition to this ATG12 also contributes to cellular responses to stress conditions via this pathway. Furthermore it has connections with signaling pathways involving proteins such as LC3 which marks autophagosomal membranes and interacts with ATG12-conjugated complexes enhancing the autophagic flux.
- Associated diseases and disorders
Researchers associate dysregulated expression of ATG12 with neurodegenerative diseases such as Parkinson’s disease where impaired autophagy leads to toxic protein accumulation. Additionally alterations in ATG12 expression have links to cancer as enhanced autophagy in some tumor cells supports survival under metabolic stress. Both disorders demonstrate connections to proteins involved in the autophagy pathway suggesting therapeutic potential in modulating ATG12 activity.
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2 product images
Western blot - Human ATG12 knockout THP-1 cell line (ab277831)
False colour image of Western blot: Anti-ATG12 antibody staining at 1/500 dilution shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-ATG12 antibody ab155589 was shown to bind specifically to ATG12. A band likely to be the unfunctional complex with ATG5 was observed at 52 kDa in wild-type THP-1 cell lysates with no signal observed at this size in Atg12 knockout cell line ab277831 (knockout cell lysate ab278183) - unconjugated functional form not observed at 15 kD. To generate this image wild-type and Atg12 knockout THP-1 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°. 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-ATG12 antibody (Anti-ATG12 antibody ab155589) at 1/500 dilution
Lane 1: Wild-type THP-1 cell lysate at 20 µg
Lane 2: ATG12 knockout THP-1 cell lysate at 20 µg
Lane 3: HCT 116 cell lysate at 20 µg
Lane 4: HeLa cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 52 kDa
Western blot - Human ATG12 knockout THP-1 cell line (ab277831)
False colour image of Western blot: Anti-ATG12 antibody [EPR4800] staining at 1/1000 dilution shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-ATG12 antibody [EPR4800] ab109491 was shown to bind specifically to ATG12. A band likely to be the unfunctional complex with ATG5 was observed at 52 kDa in wild-type THP-1 cell lysates with no signal observed at this size in Atg12 knockout cell line ab277831 (knockout cell lysate ab278183) - unconjugated functional form not observed at 15 kDa. To generate this image wild-type and Atg12 knockout THP-1 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°. 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-ATG12 antibody [EPR4800] (Anti-ATG12 antibody [EPR4800] ab109491) at 1/1000 dilution
Lane 1: Wild-type THP-1 cell lysate at 20 µg
Lane 2: ATG12 knockout THP-1 cell lysate at 20 µg
Lane 3: HCT 116 cell lysate at 20 µg
Lane 4: HeLa cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 52 kDa
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