Human OTUB1 knockout HEK-293T cell line
- Advanced Validation
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OTUB1 KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, 19 bp deletion in exon 1 and Insertion of the selection cassette 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 OTUB1, HSPC263, OTB1, OTU domain, ubiquitin aldehyde binding 1, OTU domain-containing Ubal-binding protein 1, OTU domain-containing ubiquitin aldehyde-binding protein 1, OTU-domain Ubal-binding 1, OTUB1_HUMAN, Otubain-1, Ubiquitin thioesterase OTUB1, Ubiquitin-specific-processing protease OTUB1, hOTU1, ubiquitin-specific protease otubain 1
- ICC/IF
Supplier Data
Immunocytochemistry/ Immunofluorescence - Human OTUB1 knockout HEK-293T cell line (AB266551)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized OTUB1 KO HEK293T (ab266551) cells labelling OTUB1 with ab270959 at 1/250 (2.204 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing no staining in OTUB1 KO HEK293T cell line and nuclear and cytoplasmic staining in Parental HEK293T. is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.
- WB
Lab
Western blot - Human OTUB1 knockout HEK-293T cell line (AB266551)
Blocking and diluting buffer and concentration : 5% NFDM/TBST
Lanes 1-3 : Merged signal (red and green). Green -ab270959 observed at 31kDa. Red - loading control ab8245 observed at 36 kDa. ab270959 Anti-OTUB1 antibody [EPR24917-75] was shown to specifically react with OTUB1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE.
ab270959 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-OTUB1 antibody [EPR24917-75] (<a href='/en-us/products/primary-antibodies/otub1-antibody-epr24917-75-ab270959'>ab270959</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg
Lane 2:
OTUB1 knockout HEK293T (ab266551) whole cell lysate at 20 µg
Lane 2:
Western blot - Human OTUB1 knockout HEK-293T cell line (ab266551)
Lane 3:
HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Secondary
All lanes:
Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution
Predicted band size: 31 kDa
false
- WB
Lab
Western blot - Human OTUB1 knockout HEK-293T cell line (AB266551)
Lanes 1-2 : Merged signal (red and green). Green - ab101471 observed at 130 kDa. Red - loading control ab8245 observed at 37 kDa.
ab101471 Anti-OTUB1 antibody was shown to specifically react with OTUB1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab101471 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 10000 ug/ml and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and 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-OTUB1 antibody (<a href='/en-us/products/primary-antibodies/otub1-antibody-ab101471'>ab101471</a>) at 1/10000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
OTUB1 knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human OTUB1 knockout HEK-293T cell line (ab266551)
Predicted band size: 31 kDa
Observed band size: 130 kDa
false
- WB
Lab
Western blot - Human OTUB1 knockout HEK-293T cell line (AB266551)
Lanes 1-2 : Merged signal (red and green). Green - ab175200 observed at 31 kDa. Red - loading control ab8245 observed at 37 kDa.
ab175200 Anti-OTUB1 antibody [EPR13028(B)] was shown to specifically react with OTUB1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab175200 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 Dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and 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-OTUB1 antibody [EPR13028(B)] (<a href='/en-us/products/primary-antibodies/otub1-antibody-epr13028b-ab175200'>ab175200</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
OTUB1 knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human OTUB1 knockout HEK-293T cell line (ab266551)
Predicted band size: 31 kDa
Observed band size: 130 kDa,31 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human OTUB1 knockout HEK-293T cell line (AB266551)
Allele-2 : Insertion of the selection cassette in exon 1.
- Sanger seq
Unknown
Sanger Sequencing - Human OTUB1 knockout HEK-293T cell line (AB266551)
Allele-1 : 19 bp deletion in exon 1
- Cell Culture
Unknown
Cell Culture - Human OTUB1 knockout HEK-293T cell line (AB266551)
Representative images of OTUB1 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 XL Core microscope.
Reactivity data
Product details
We will provide viable cells that proliferate on revival.
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
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
OTUB1 functions as a deubiquitinating enzyme that cleaves ubiquitin from substrates influencing various cellular processes. It acts independently or as part of larger protein complexes. By controlling protein degradation pathways OTUB1 impacts protein turnover and cellular homeostasis. Interactions with other proteins allow OTUB1 to exert influence on different signaling pathways and cellular stress responses.
Pathways
OTUB1 holds a significant position in the regulation of the ubiquitin-proteasome system and DNA damage repair pathway. It closely interacts with proteins such as UBE2N affecting the DNA damage response. Additionally OTUB1 regulates processes through modulating pathways like NF-kB by interacting with and stabilizing specific protein factors involved.
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
Complementary Products
Primary Antibodies
AB175200
Anti-OTUB1 antibody [EPR13028(B)]
RabMAb|Recombinant|KO Validated|20ul selling size
![Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] (AB175200)](https://content.abcam.com/products/images/otub1-antibody-epr13028b-ab175200--immunoprecipitation-img278.jpg)
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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