Anti-Smad4 antibody [EP618Y](AB40759)

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:

Western blot - Anti-Smad4 antibody [EP618Y] (ab40759)

Predicted band size: 60 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

All lanes:

Western blot - Anti-Smad4 antibody [EP618Y] (ab40759) at 1/5000 dilution

Lane 1:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) cell lysate at 10 µg

Lane 2:

Ramos (Human Burkitt's lymphoma cell line) cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 60 kDa

Observed band size: 60 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.

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes:

Western blot - Anti-Smad4 antibody [EP618Y] (ab40759) at 1/5000 dilution

Lane 1:

NIH/3T3 (Mouse embyro fibroblast cell line) cell lysate at 20 µg

Lane 2:

Mouse embryo tissue lysate at 20 µg

Lane 3:

Mouse skin tissue lysate at 20 µg

Lane 4:

Mouse lung tissue lysate at 20 µg

Lane 5:

PC-12 (Rat adrenal gland pheochromocytoma cell line) cell lysate at 20 µg

Lane 6:

C6 cell lysate at 20 µg

Lane 7:

Rat skin tissue lysate at 20 µg

Lane 8:

Rat lung tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 60 kDa

Observed band size: 60 kDa

Exposure time: 3min

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes:

Western blot - Anti-Smad4 antibody [EP618Y] (ab40759) at 1/5000 dilution

Lane 1:

SW480 (Human colorectal adenocarcinoma cell line) cell lysate at 20 µg

Lane 2:

HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate at 20 µg

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate at 20 µg

Lane 4:

Human skin tissue lysate at 20 µg

Lane 5:

Human lung tissue lysate at 20 µg

Lane 6:

Human artery tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 60 kDa

Observed band size: 60 kDa

Exposure time: 30s

Different batches of ab40759 were tested on Ramos (Human Burkitt's lymphoma B lymphocyte) lysate at 0.7 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 60 kDa.

All lanes:

Western blot - Anti-Smad4 antibody [EP618Y] (ab40759)

Predicted band size: 60 kDa