All lanes : Anti-Smad2 antibody [EP784Y] (ab40855) at 1/2000 dilution

Lane 1 : A549 cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : SMAD2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 58 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab40855 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab40855 was shown to react with Smad2 in wild-type HeLa. Loss of signal was observed when knockout cell line ab255430 (knockout cell lysate ab263833) was used. Wild-type and Smad2 knockout samples were subjected to SDS-PAGE. ab40855 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 dilution and 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.

 

All lanes : Anti-Smad2 antibody [EP784Y] (ab40855) at 1/1000 dilution

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : Smad2 knockout HeLa whole cell lysate
Lane 3 : A549 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 58 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab40855 observed at 58 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab40855 was shown to specifically react with Smad2 in wild-type HeLa cells as signal was lost in Smad2 knockout cells. Wild-type and SMAD2 knockout samples were subjected to SDS-PAGE. Ab40855 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 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-Smad2 antibody [EP784Y] (ab40855) at 1/2000 dilution

Lane 1 : A-673 (Human muscle Ewing's Sarcoma cell line) whole cell lysate
Lane 2 : HUVEC (Human umbilical vein endothelial cell line) whole cell lysate
Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate

Lysates/proteins at 20 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: 58 kDa

Diluting and blocking buffer: 5% NFDM /TBST

ab40855 staining Smad2 in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab195889 was used as a counterstain for primary antibody ab40855 at 1/1000. DAPI was used as a nuclear counterstain and PBS as a negative control.

All lanes : Anti-Smad2 antibody [EP784Y] (ab40855) at 1/10000 dilution

Lane 1 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lanes 2-3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 20000 µg (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 58 kDa

Blocking and diluting buffer: 5% NFDM/TBST

ab40855 stainingSmad2 in human bladder carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/50. A ImmunoHistoProbe one step HRP Polymer was used as a secondary antibody, ready to use.

Negative control 1: PBS in place of primary antibody.

ab40855 (purified) at 1/20 immunoprecipitating EGFR in HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate.

Lane 1 (input): HeLa whole cell lysate (10µg)

Lane 2 (+): ab40855 + HeLa whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40855 in HeLa whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/1000).

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

ab40855 staining Smad2 in the human cell line HeLa (Human epithelial cell line from cervix adenocarcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

Immunofluorescence staining of HeLa cells with purified ab40855 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was an Alexa Fluor^® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.

Overlay histogram showing PC3 cells stained with ab40855 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab40855, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Anti-Smad2 antibody [EP784Y] (ab40855) at 1/500000 dilution + Jurkat cell lysate

Predicted band size: 58 kDa
Observed band size: 58 kDa

ab40855 at a 1:100 dilution staining Smad2 in human prostate carcinoma tissue.