SQSTM1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 4.
Images
Key facts
- Cell type
- HEK-293T
- Species or organism
- Human
- Tissue
- Kidney
- Form
- Liquid
- Knockout validation
- Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 4.
Alternative names
A170, DMRV, EBI 3 associated protein p60, EBI3-associated protein of 60 kDa, EBIAP, FTDALS3, MGC127197, ORCA, OSF-6, OSIL, Osi, Oxidative stress induced like, P60, PDB 3, PKC-zeta-interacting protein, Paget disease of bone 3, Phosphotyrosine independent ligand for the Lck SH2 domain p62, Phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa, Protein kinase C-zeta-interacting protein, SQSTM 1, SQSTM_HUMAN, STAP, STONE14, Sequestosome-1, Ubiquitin-binding protein p62, ZIP, ZIP 3, p62, p62B
Recommended products
SQSTM1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 4.
Key facts
- Cell type
- HEK-293T
- Species or organism
- Human
- Tissue
- Kidney
- Form
- Liquid
- Knockout validation
- Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 4.
- Concentration
Properties
- Gene name
- SQSTM1
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Sanger Sequencing, Western blot
- Zygosity
- Homozygous
Quality control
- STR analysis
- D7S820, D5S818, TH01, D16S539, TPOX, CSF1PO, D13S317
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
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.
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4 product images
Western blot - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
Lanes 1- 2: Merged signal (red and green). Green - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free ab56416 observed at 62 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) observed at 37 kDa.
Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free ab56416 was shown to react with SQSTM1 in wild-type HEK293Tcells in western blot. Loss of signal was observed when knockout cell line ab255343 (knockout cell lysate Human SQSTM1 (p62) knockout HEK-293T cell lysate ab263770) was used. Wild-type HEK293Tand SQSTM1 knockout HEK293Tcell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free ab56416 and Anti-GAPDH antibody[EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye®800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat Anti-Rabbit IgG H&L (IRDye®680RD) preadsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free ab56416) at 1/1000 dilution
Lane 1: Wild-type HEK293T cell lysate at 20 µg
Lane 2: SQSTM1 knockout HEK293T cell lysate at 20 µg
Lane 2: Western blot - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 62 kDa
Western blot - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
Lanes 1- 2: Merged signal (red and green). Green - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012 observed at 64 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012 was shown to react with SQSTM1 in wild-type HEK293Tcells in western blot. Loss of signal was observed when knockout cell line ab255343 (knockout cell lysate Human SQSTM1 (p62) knockout HEK-293T cell lysate ab263770) was used. Wild-type HEK293Tand SQSTM1 knockout HEK293Tcell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 10000 Dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012) at 1/10000 dilution
Lane 1: Wild-type HEK293T cell lysate at 20 µg
Lane 2: SQSTM1 knockout HEK293T cell lysate at 20 µg
Lane 2: Western blot - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 64 kDa
Sanger Sequencing - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
Sequencing chromatogram displaying sequence edit in exon 4
Sanger Sequencing - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
Homozygous: 1 bp insertion in exon 4
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