Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: SMAD4 knockout  HAP1 whole cell lysate (20 µg)
Lane 3: HepG2 whole cell lysate (20 µg)
Lane 4: Jurkat whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab40759 observed at 60 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab40759 was shown to specifically react with SMAD4 in wild type HAP1 cells. No band was observed when SMAD4 knockout HAP1 samples were used. Wild-type and SMAD4 knockout samples were subjected to SDS-PAGE. Ab40759 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution 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 [EP618Y] (ab40759) at 1/5000 dilution (purified)

Lane 1 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) cell lysate
Lane 2 : Ramos (Human Burkitt's lymphoma cell line) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 65 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Smad4 with purified ab40759 at 1/100. 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.

All lanes : Anti-Smad4 antibody [EP618Y] (ab40759) at 1/5000 dilution

Lane 1 : NIH/3T3 (Mouse embyro fibroblast cell line) cell lysate
Lane 2 : Mouse embryo tissue lysate
Lane 3 : Mouse skin tissue lysate
Lane 4 : Mouse lung tissue lysate
Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma cell line) cell lysate
Lane 6 : C6 cell lysate
Lane 7 : Rat skin tissue lysate
Lane 8 : Rat lung 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: 65 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Smad4 antibody [EP618Y] (ab40759) at 1/5000 dilution

Lane 1 : SW480 (Human colorectal adenocarcinoma cell line) cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate
Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) 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: 65 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

Anti-Smad4 antibody [EP618Y] (ab40759) at 1/10000 dilution (purified) + NIH/3T3 (Mouse embyro fibroblast cell line) cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 65 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST.

Anti-Smad4 antibody [EP618Y] (ab40759) at 1/5000 dilution (purified) + PC-12 (Rat adrenal gland pheochromocytoma cell line) cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 65 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST.

Anti-Smad4 antibody [EP618Y] (ab40759) at 1/5000 dilution (unpurified) + SHSY5Y (Human neuroblastoma cell line from bone marrow) cell lysate at 10 µg

Predicted band size: 65 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue labelling Smad4 with unpurified ab40759 at a 1/100 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Unpurified ab40759 staining Smad4 in rat femur tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 20 minutes at 22°C; antigen retrieval was by heat mediation in a citrate buffer pH6.0. Samples were incubated with primary antibody (1/200 in blocking buffer) for 2 hours at 20°C. An undiluted HRP-conjugated goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

See Abreview