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A synthetic peptide corresponding to residues near the N term of TRAF6 (Human)
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents
This product is a recombinant rabbit monoclonal antibody.
Our Abpromise guarantee covers the use of ab33915 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/2000 - 1/10000. Predicted molecular weight: 58 kDa.Can be blocked with TRAF6 peptide (ab183540).|
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
|IHC-P||Use at an assay dependent concentration.|
Overlay histogram showing HAP1 wildtype (green line) and HAP1-TRAF6 knockout cells (red line) stained with ab33915. The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab33915, 0.1µg/ml ) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) preadsorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.
A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-TRAF6 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.
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 - ab33915 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.
Ab33915 was shown to specifically react with TRAF6 in wild-type cells as signal was lost in TRAF6 knockout HAP1 cells. Wild-type and TRAF6 knockout samples were subjected to SDS-PAGE. Ab33915 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 2000 dilution 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
ab33915 staining TRAF6 in Human platelet cells by Flow cytometry.
Cells were fixed in paraformaldehyde and permeabilized using 0.1% Triton-X-100 in 2% BSA for 15 minutes. Primary antibody used at a 1/200 dilution and incubated for 16 hours at 4°C. The secondary antibody used was an Alexa Fluor®488 conjugated chicken anti-rabbit IgG (H+L) at a 1/500 dilution.
Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling TRAF6 with purified ab33915 at 1/240 dilution(10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.
This antibody is unsuitable for detecting tissue lysates.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"