All lanes : Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution

Lane 1 : Wild-type 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 - ab33875 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.

 ab33875 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. ab33875 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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 [EP567Y] (ab33875) 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
Observed band size: 52 kDa why is the actual band size different from the predicted?

Lanes 1 - 3: Merged signal (red and green). Green - ab33875 observed at 52 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab33875 was shown to specifically react with Smad2 in wild-type WT HeLa cells as signal was lost in SMAD2 knockout cells. Wild-type and SMAD2 knockout samples were subjected to SDS-PAGE. Ab33875 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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.

Immunocytochemistry/Immunofluorescent analysis of HeLa (human cervix adenocarcinoma epithelial) cells labeling Smad2 with ab33875 at a dilution of 1/500. ab150077, an Alexa Fluor^® 488 goat anti-rabbit was used at 1/1000 was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Counterstain antibody: ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200.

Secondary antibody only negative control is shown in the bottom panels.

Confocal image showing mainly nuclear staining on HeLa cells after the treatment with TGF-b (10ng/mL) for 1 hour.

Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution (Purified) + RAW264.7 at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-Smad2 antibody [EP567Y] (ab33875) at 1/2000 dilution (Purified) + Jurkat cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Overlay histogram showing Jurkat cells stained with purified ab33875 (pink line) at a dilution of 1/110. The cells were fixed with 2% PFA. FITC goat anti-rabbit was used at a dilution of 1/150 and rabbit monoclonal IgG was used as the isotype control (green line).

Anti-Smad2 antibody [EP567Y] (ab33875) at 1/500 dilution + RAW264.7 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 58 kDa
Additional bands at: 58 kDa. We are unsure as to the identity of these extra bands.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution (Unpurified) + Jurkat cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 58 kDa
Additional bands at: 58 kDa. We are unsure as to the identity of these extra bands.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Overlay histogram showing PC3 cells stained with unpurified ab33875 (red line). The cells were fixed with 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 (ab33875, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in PC3 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.