Human TRAF6 knockout HeLa cell line (ab266009)
Overview
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Product name
Human TRAF6 knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HeLa cell line (ab255928). 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.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80ºC. Storage at -80ºC may result in loss of viability.
1. Thaw the vial in 37ºC water bath 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 (Click here to view haemocytometer protocol) or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. This should allow for confluency within 48 hours. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37ºC incubator with 5% CO2. 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 for confluency (80-90% confluence) within 48 hours.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
Click here to view the Mammalian cell tissue culture protocol
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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~90% -
Adherent /Suspension
Adherent -
Tissue
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10 -
Antibiotic resistance
Puromycin 1.00µg/ml -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% DMSO, 2% Cellulose, methyl ether -
Research areas
Target
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Function
E3 ubiquitin ligase that, together with UBE2N and UBE2V1, mediates the synthesis of 'Lys-63'-linked-polyubiquitin chains conjugated to proteins, such as IKBKG, AKT1 and AKT2. Also mediates ubiquitination of free/unanchored polyubiquitin chain that leads to MAP3K7 activation. Leads to the activation of NF-kappa-B and JUN. May be essential for the formation of functional osteoclasts. Seems to also play a role in dendritic cells (DCs) maturation and/or activation. Represses c-Myb-mediated transactivation, in B lymphocytes. Adapter protein that seems to play a role in signal transduction initiated via TNF receptor, IL-1 receptor and IL-17 receptor. -
Tissue specificity
Expressed in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. -
Pathway
Protein modification; protein ubiquitination. -
Sequence similarities
Belongs to the TNF receptor-associated factor family. A subfamily.
Contains 1 MATH domain.
Contains 1 RING-type zinc finger.
Contains 2 TRAF-type zinc fingers. -
Domain
The coiled coil domain mediates homo- and hetero-oligomerization.
The MATH/TRAF domain binds to receptor cytoplasmic domains. -
Post-translational
modificationsSumoylated on Lys-124, Lys-142 and Lys-453 by SUMO1.
Polyubiquitinated on Lys-124; after cell stimulation with IL-1-beta or TGF-beta. This ligand-induced cell stimulation leads to dimerization/oligomerization of TRAF6 molecules, followed by auto-ubiquitination which involves UBE2N and UBE2V1 and leads to TRAF6 activation. This 'Lys-63' site-specific poly-ubiquitination appears to be associated with the activation of signaling molecules. Endogenous autoubiquitination occurs only for the cytoplasmic form. -
Cellular localization
Cytoplasm. Cytoplasm > cell cortex. Nucleus. Found in the nuclei of some agressive B-cell lymphoma cell lines as well as in the nuclei of both resting and activated T-and B-lymphocytes. Found in punctate nuclear body protein complexes. Ubiquitination may occur in the cytoplasm and sumoylation in the nucleus. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
Our Abpromise guarantee covers the use of ab266009 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB | Use at an assay dependent concentration. Predicted molecular weight: 60 kDa. |
Images
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All lanes : Anti-TRAF6 antibody [EP592Y] (ab40675) at 1/500 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TRAF6 knockout HeLa cell lysate
Lane 3 : HAP1 cell lysate
Lane 4 : Daudi cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 60 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?Lanes 1- 4: Merged signal (red and green). Green - ab40675 observed at 65 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab40675 was shown to react with TRAF6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab266009 (knockout cell lysate ab257760) was used. Wild-type HeLa and TRAF6 knockout HeLa cell 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. ab40675 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 500 dilution and a 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.
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All lanes : Anti-TRAF6 antibody [EP591Y] (ab33915) at 1/2000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TRAF6 knockout HeLa cell lysate
Lane 3 : HAP1 cell lysate
Lane 4 : Daudi cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 60 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?Lanes 1- 4: Merged signal (red and green). Green - ab33915 observed at 65 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab33915 was shown to react with TRAF6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab266009 (knockout cell lysate ab257760) was used. Wild-type HeLa and TRAF6 knockout HeLa cell 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. ab33915 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 dilution and a 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.
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Homozygous: 1 bp insertion in exon 2.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
References (0)
ab266009 has not yet been referenced specifically in any publications.