This antibody gave a positive signal in the following tissue lysates: Human lung; Human kidney; Human heart; Human placenta; Human skeletal muscle as well as the following whole cell lysates: HeLa; Jurkat; K562; PC3; NIH3T3.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 60 kDa (predicted molecular weight: 60 kDa).
Use a concentration of 1 µg/ml.
Use a concentration of 5 µg/ml.
FunctionE3 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 specificityExpressed in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.
PathwayProtein modification; protein ubiquitination.
Sequence similaritiesBelongs 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.
DomainThe 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 localizationCytoplasm. 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.
TNF receptor-associated factor 6, E3 ubiquitin protein ligase antibody
TRAF 6 antibody
Anti-TRAF6 antibody images
Western blot - Anti-TRAF6 antibody (ab94720)
Predicted band size : 60 kDa
Lane 1: Wild type HAP1 whole cell lysate (20 µg) Lane 2: TRAF6 knockout HAP1 whole cell lysate (20 µg) Lane 3: HeLa whole cell lysate (20 µg) Lane 4: HEK293 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab94720 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab94720 was shown not to specifically react with TRAF6 when TRAF6 knockout samples were used. Wild-type and TRAF6 knockout samples were subjected to SDS-PAGE. Ab94720 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
TRAF6 was immunoprecipitated using 0.5mg Mouse Skeletal Muscle tissue lysate, 5µg of Rabbit polyclonal to TRAF6 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Skeletal Muscle tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab94720.
All lanes : Anti-TRAF6 antibody (ab94720) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 3 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate Lane 4 : PC3 (Human prostate carcinoma cell line) Whole Cell Lysate Whole Cell Lysate Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 6 : Lung (Human) Tissue Lysate Lane 7 : Human kidney tissue lysate - total protein (ab30203) Lane 8 : Human heart tissue lysate - total protein (ab29431) Lane 9 : Human placenta tissue lysate - total protein (ab29745) Lane 10 : Human skeletal muscle tissue lysate - total protein (ab29330)
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 60 kDa Observed band size : 60 kDa Additional bands at : 100 kDa. We are unsure as to the identity of these extra bands.
ICC/IF image of ab94720 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab94720 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in formaldehyde fixed (4%, 10min) MCF-7 cell types at 1ug/ml
References for Anti-TRAF6 antibody (ab94720)
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