Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: TAK1 knockout HAP1 cell lysate (20 µg)
Lane 3: SHSY5Y cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab109526 observed at 72 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109526 was shown to specifically react with TAK1 when TAK1 knockout samples were used. Wild-type and TAK1 knockout samples were subjected to SDS-PAGE. ab109526 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

ab109526 staining TAK1 in wild-type HAP1 cells (top panel) and TAK1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109526 at 1/1000 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Flow Cytometry analysis of A431(human epidermoid carcinoma) cells labeling TAK1 with unpurified ab109526 at 1/20 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-TAK1 antibody [EPR5984] (ab109526) at 1/1000 dilution

Lane 1 : K562 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : A431 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

Predicted band size: 67 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

All lanes : Anti-TAK1 antibody [EPR5984] (ab109526) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : MAP3K7 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 67 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab109526 observed at 72 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109526 was shown to react with TAK1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266555 (knockout cell lysate ab256984) was used. Wild-type HEK-293T and MAP3K7 knockout HEK-293T 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. ab109526 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 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.

ab109526, at a 1/50 dilution, staining TAK1 in Formalin/PFA-fixed paraffin-embedded Human brain tissue, by Immunohistochemistry.

Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.