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: 58 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.

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.

Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using anti-TRAF6 (ab33915)

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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.

P : Permeabilized
US : Unstained (Red Peak) IGG RB : IgG Rabbit (Blue Peak)
TRAF6 Ab (Green Peak)

See Abreview

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

Anti-TRAF6 antibody [EP591Y] (ab33915) at 1/10000 dilution + Jurkat cell lysate

Predicted band size: 58 kDa

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.

All lanes : Anti-TRAF6 antibody [EP591Y] (ab33915) at 1/1000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Human heart lysates
Lane 3 : Human skeletal muscle lysates
Lane 4 : Mouse skeletal muscle lysates
Lane 5 : Rat skeletal muscle lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 58 kDa

This antibody is unsuitable for detecting tissue lysates.

All lanes : Anti-TRAF6 antibody [EP591Y] (ab33915) at 1/1000 dilution

Lane 1 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 58 kDa