Human USP14 (TGT) knockout HeLa cell line
- Advanced Validation
- What is this?
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USP14 KO cell line available to order. KO validated. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 1. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
View Alternative Names
Deubiquitinating enzyme 14, UBP14_HUMAN, USP 14, Ubiquitin carboxyl-terminal hydrolase 14, Ubiquitin specific peptidase 14, Ubiquitin specific protease 14, Ubiquitin thiolesterase 14, Ubiquitin-specific-processing protease 14, tRNA guanine transglycosylase 60 kD subunit, tRNA-guanine transglycosylase
- WB
Lab
Western blot - Human USP14 (TGT) knockout HeLa cell line (AB266854)
False colour image of Western blot : Anti-USP14/TGT antibody [EPR15943] - C-terminal staining at 1/1000 dilution shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution shown in red. In Western blot ab192618 was shown to bind specifically to USP14/TGT. A band was observed at 63 kDa in wild-type HeLa cell lysates with no signal observed at this size in USP14 CRISPR-Cas9 edited cell line ab266854 (CRISPR-Cas9 edited cell lysate ab257787). The band observed in the CRISPR-Cas9 edited lysate lane below 63 kDa is likely to represent a truncated form of USP14/TGT. 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 USP14 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.
All lanes:
Western blot - Anti-USP14/TGT antibody [EPR15943] - C-terminal (<a href='/en-us/products/primary-antibodies/usp14-tgt-antibody-epr15943-c-terminal-ab192618'>ab192618</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
USP14 CRISPR-Cas9 edited HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human USP14 (TGT) knockout HeLa cell line (ab266854)
Lane 3:
USP14 knockout HeLa cell lysate at 20 µg
Predicted band size: 56 kDa
Observed band size: 63 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human USP14 (TGT) knockout HeLa cell line (AB266854)
Homozygous : 1 bp insertion in exon 1
Reactivity data
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
USP14 functions in regulating protein turnover by removing ubiquitin from substrate proteins impacting their stability and degradation. USP14 is part of a larger protein complex associated with the proteasome specifically binding to the 19S regulatory particle. It is important in maintaining cellular protein homeostasis by editing ubiquitin chains on target proteins prior to degradation.
Pathways
USP14 participates in ubiquitin-proteasome pathways and ER-associated degradation pathway. In these processes it works closely with other proteasome proteins such as Rpn11 which also removes ubiquitin from proteins. The regulation of these pathways by USP14 directly affects protein quality control in cells influencing processes like cell cycle and stress response.
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
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Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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