All lanes : Anti-Smad4 antibody (ab110175) at 1 µg

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : SMAD4 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 60 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab110175 observed at 60 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab110175 was shown to recognize Smad4 in wild-type HAP1 cells as signal was lost at the expected MW in SMAD4 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SMAD4 knockout samples were subjected to SDS-PAGE. Ab110175 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Smad4 antibody (ab110175) at 1/1000 dilution

Lane 1 : SW480 cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Human skin tissue lysate
Lane 5 : Human lung tissue lysate
Lane 6 : Human artery tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 60 kDa
Observed band size: 60 kDa


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Smad4 antibody (ab110175) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 4 : A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate
Lane 5 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 6 : THP1 (Human acute monocytic leukemia cell line) Whole Cell Lysate
Lane 7 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : 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: 67 kDa why is the actual band size different from the predicted?
Additional bands at: 37 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes


The predicted molecular weight of Smad4 is 60 kDa (SwissProt), however we expect to observe a banding pattern around 70 kDa. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.