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: 63 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebral cortex tissue labelling TRAF6 with purified ab40675 at 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling TRAF6 with purified ab40675 at 1/50. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/50) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue labelling TRAF6 with purified ab40675 at 1/50. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling TRAF6 with unpurified ab40675 at 1/250.

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 - ab40675 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.

 

Ab40675 was shown to specifically react with TRAF6 in wild-type cells, along with additional cross-reactive bands as signal was lost in TRAF6 knockout HAP1 cells. Wild-type and TRAF6 knockout samples were subjected to SDS-PAGE. Ab40675 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 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.

All lanes : Anti-TRAF6 antibody [EP592Y] (ab40675) at 1/5000 dilution (purified)

Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 63 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-TRAF6 antibody [EP592Y] (ab40675) at 1/1000 dilution (purified) + HEK293 cell lysate at 20 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 63 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

 

Anti-TRAF6 antibody [EP592Y] (ab40675) at 1/2000 dilution (unpurified) + 10ug Jurkat cell lysate

Predicted band size: 63 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?

All lanes : Anti-TRAF6 antibody [EP592Y] (ab40675) 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: 63 kDa


Exposure time: 70 seconds

This antibody is unsuitable for detecting tissue lysates.  

All lanes : Anti-TRAF6 antibody [EP592Y] (ab40675) 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: 63 kDa