PINK1 KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 10 bp deletion in exon 1.
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- Journal of dairy science 105:7829-78412022Low abundance of mitophagy markers is associated with reactive oxygen species overproduction in cows with fatty liver and causes reactive oxygen species overproduction and lipid accumulation in calf hepatocytes.Applications:Unspecified applicationReactive species:Unspecified reactive speciesZhiyuan Fang,Guowen Liu,Mengyao Zhu,Shu Wang,Qianming Jiang,Juan J Loor,Hao Yu,Xue Hao,Meng Chen,Wenwen Gao,Lin Lei,Yuxiang Song,Zhe Wang,Xiliang Du,Xinwei LiPubMed 35863923
Key facts
- Cell type
- HEK-293T
- Species or organism
- Human
- Tissue
- Kidney
- Form
- Liquid
- Knockout validation
- Sanger Sequencing
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 10 bp deletion in exon 1
Alternative names
BRPK, FLJ27236, PARK 6, PINK1_HUMAN, PTEN induced putative kinase 1, PTEN-induced putative kinase protein 1, Phosphatase and Tensin Homolog, Protein kinase BRPK, Serine/threonine kinase PINK1 mitochondrial, Serine/threonine protein kinase PINK1 mitochondrial, Serine/threonine-protein kinase PINK1, mitochondrial
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PINK1 KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 10 bp deletion in exon 1.
Key facts
- Cell type
- HEK-293T
- Species or organism
- Human
- Tissue
- Kidney
- Form
- Liquid
- Knockout validation
- Sanger Sequencing
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 10 bp deletion in exon 1
- Concentration
Properties
- Gene name
- PINK1
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Sanger Sequencing
- Zygosity
- Homozygous
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 2 US: 2
- Adherent/suspension
- Adherent
- Gender
- Female
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.
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- -196°C
- Appropriate long-term storage conditions
- -196°C
Notes
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
The PTEN-induced kinase 1 (PINK1) is a serine/threonine-protein kinase with a molecular weight of approximately 63 kDa. It plays a significant role in mitochondrial quality control through its kinase activity. PINK1 gets expressed in various tissues with a high presence in the brain. The protein localizes to the outer membrane of damaged mitochondria to initiate mitophagy an important cellular process for clearing dysfunctional mitochondria.
- Biological function summary
The PINK1 protein detects mitochondrial damage and recruits Parkin an E3 ubiquitin ligase to the damaged mitochondria. This interaction leads to the ubiquitylation of mitochondrial substrates and triggers their degradation. PINK1 Parkin and other proteins form a complex that facilitates the removal of damaged mitochondria through autophagy. This mechanism ensures cellular health and energy balance by maintaining a pool of functional mitochondria.
- Pathways
PINK1 integrates into the mitochondrial quality control and mitophagy pathways. It has a fundamental role in the PINK1-Parkin pathway which is critical for maintaining mitochondrial integrity. In this pathway PINK1 phosphorylates both ubiquitin and Parkin enhancing Parkin’s E3 ligase activity. Another pathway that PINK1 participates in is the PTEN-induced kinase pathway where it regulates mitochondrial dynamics and homeostasis via cross-talk with other mitochondrial proteins such as DJ-1 and LRRK2.
- Associated diseases and disorders
PINK1 mutations are strongly associated with familial Parkinson’s disease attributing to its role in preserving neuronal function through mitochondrial regulation. Deficient PINK1 function leads to accumulation of damaged mitochondria contributing to neuronal cell death. Additionally PINK1 dysfunction is implicated in Alzheimer’s disease as impaired mitochondrial clearance is a contributing factor. Here the PINK1 interaction with other proteins like Parkin and DJ-1 highlights its importance in neurodegenerative disorders.
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7 product images
Cell Culture - Human PINK1 knockout HEK-293T cell line (ab266393)
Representative images PINK1 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS M5000 microscope.
Western blot - Human PINK1 knockout HEK-293T cell line (ab266393)
False colour image of Western blot: Anti-PINK1 antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to PINK1. A band was observed at 63 kDa in wild-type cell lysates with no signal observed at this size in PINK1 knockout cell line ab266393 (knockout cell lysate ab257030). To generate this image, wild-type and PINK1 knockout 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 (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: Anti-PINK1 antibody at 1/1000 dilution
Lane 1: Wild-type HEK-293T Vehicle Control CCCP (0 μM, 24 h) cell lysate at 20 µg
Lane 2: Western blot - Human PINK1 knockout HEK-293T cell line (ab266393)
Lane 2: Wild-type HEK-293T Treated CCCP (10 μM, 24 h) cell lysate at 20 µg
Lane 3: PINK1 knockout HEK-293T Vehicle Control CCCP (0 μM, 24 h) cell lysate at 20 µg
Lane 4: PINK1 knockout HEK-293T Treated CCCP (10 μM, 24 h) cell lysate at 20 µg
Lane 5: HeLa cell lysate at 20 µg
Lane 6: Daudi cell lysate at 20 µg
SecondaryLanes 1 - 6: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution
Lanes 1 - 6: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 63 kDa
Western blot - Human PINK1 knockout HEK-293T cell line (ab266393)
Anti-PINK1 antibody [MJF-R32-7] (Anti-PINK1 antibody [MJF-R32-7] ab300623) staining at 1/1000 dilution, shown in black; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. Anti-PINK1 antibody [MJF-R32-7] ab300623 was shown to bind specifically to PINK1. A band was observed at 60 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in PINK1 knockout cell line ab266393 (knockout cell lysate ab257030). To generate this image, wild-type and PINK1 knockout HEK-293 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 before development with Optiblot (ECL reagent Anti-PKC delta (phospho S299) antibody [EPNCI119] ab133456) and imaged with 4 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-PINK1 antibody [MJF-R32-7] (Anti-PINK1 antibody [MJF-R32-7] ab300623) at 1/1000 dilution
Lane 1: Wild-type HEK-293 Vehicle Control CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (0 µM, 24 h) cell lysate at 20 µg
Lane 2: Wild-type HEK-293 Treated CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (10 µM, 24 h) cell lysate at 20 µg
Lane 3: PINK1 knockout HEK-293 Vehicle Control CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (0 µM, 24 h) cell lysate at 20 µg
Lanes 3 - 4: Western blot - Human PINK1 knockout HEK-293T cell line (ab266393)
Lane 4: PINK1 knockout HEK-293 Treated CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (10 µM, 24 h) cell lysate at 20 µg
SecondaryAll lanes: HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 63 kDa
Immunocytochemistry/ Immunofluorescence - Human PINK1 knockout HEK-293T cell line (ab266393)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PINK1 KO HEK-293T (PINK1 knockout human embryonic kidney epithelial cell), ab266393 cells labelling PINK1 with Anti-PINK1 antibody [EPR29146-340] ab323807 at 1/50 (10.34 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 g/ml dilution (Green).
Confocal image showing increased mitochondrial staining in wildtype HEK-293T cells treated with 10 μM Valinomycin for 24 hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 g/ml dilution.
anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain tubulin at 1/200 2.5 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).
Counterstain Secondary antibody only control: Secondary antibody is Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 2 g/ml dilution.
Sanger Sequencing - Human PINK1 knockout HEK-293T cell line (ab266393)
Homozygous: 10 bp deletion in exon1
Sanger Sequencing - Human PINK1 knockout HEK-293T cell line (ab266393)
Sequencing chromatogram displaying sequence edit in exon 1
Western blot - Human PINK1 knockout HEK-293T cell line (ab266393)
Anti-PINK1 antibody [EPR20730] (Anti-PINK1 antibody [EPR20730] ab216144) staining at 1/200 dilution shown in black; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. Anti-PINK1 antibody [EPR20730] ab216144 was shown to bind specifically to PINK1. A band was observed at 60 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in PINK1 knockout cell line ab266393 (knockout cell lysate ab257030). Membranes were blocked in 5 % 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 and incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with Optiblot (ECL reagent Anti-PKC delta (phospho S299) antibody [EPNCI119] ab133456) and imaged with 20 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-PINK1 antibody [EPR20730] (Anti-PINK1 antibody [EPR20730] ab216144) at 1/200 dilution
Lane 1: Wild-type HEK-293 Vehicle Control CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (0 µM, 24h) cell lysate at 20 µg
Lane 2: Wild-type HEK-293 Treated CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (10 µM, 24 h) cell lysate at 20 µg
Lanes 3 - 4: Western blot - Human PINK1 knockout HEK-293T cell line (ab266393)
Lane 3: PINK1 knockout HEK-293 Vehicle Control CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (0 µM, 24 h) cell lysate at 20 µg
Lane 4: PINK1 knockout HEK-293 Treated CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (10 µM, 24 h) cell lysate at 20 µg
SecondaryAll lanes: HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 63 kDa
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