All lanes : Anti-Smad3 antibody [EP568Y] (ab40854) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : SMAD3 knockout A549 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Human Kidney cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 48 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab40854).

Lanes 1 - 4: Merged signal (red and green). Green - ab40854 observed at 48 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab40854 was shown to react with Smad3 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab40854 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

Clone EP568Y (ab157372) has been successfully conjugated by Abcam. This image was generated using Anti-Smad3 antibody [EP568Y] (Alexa Fluor® 488). Please refer to ab204257 for protocol details.

ab204257 staining Smad3 in HepG2 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab204257 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in HepG2 cells fixed with 100% methanol (5 min).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Smad3 with purified ab40854 at 1/2000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

Flow cytometry analysis of HT-29 cells labelling Smad3 with purified ab40854 at 1/210 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

Clone EP568Y (ab157372) has been successfully conjugated by Abcam. This image was generated using Anti-Smad3 antibody [EP568Y] (Alexa Fluor® 647). Please refer to ab204461 for protocol details.

ab204461 staining Smad3 in HepG2 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab204461 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in HepG2 cells fixed with 100% methanol (5 min).

Clone EP568Y (ab157372) has been successfully conjugated by Abcam. This image was generated using Anti-Smad3 antibody [EP568Y] (PE). Please refer to ab208751 for protocol details.

ab208751 staining Smad3 in HepG2 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab208751 at 1/1000 dilution (pseudocolored in green) and ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunocytochemsitry/Immunofluorescence analysis of HepG2 cells labelling Smad3 (green) with purified ab40854 at 1/2000. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostate carcinoma tissue labelling unpurified ab40854 at 1/100 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling unpurified ab40854.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric adenocarcinoma tissue labelling unpurified ab40854.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue labelling unpurified ab40854.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung adenocarcinoma tissue labelling unpurified ab40854.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

Overlay histogram showing HCT116 cells stained with unpurified ab40854 (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 (ab40854, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40854).

This ELISA data was generated using the same anti-Smad3 antibody clone, EP568Y, in a different buffer formulation (cat# ab40854).

Standard Curve for Smad3 (Analyte: Smad3 protein (His tag) (ab89353, unpurified)); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [AF9F7] to Smad3 (ab75512) at 5µg/ml and Detector Antibody Rabbit monoclonal [EP568Y] to Smad3 (ab40854) at 0.5µg/ml.

This IHC data was generated using the same anti-Smad3 antibody clone, EP568Y, in a different buffer formulation (cat# ab40854).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic adenocarcinoma tissue labelling Smad3 with unpurified ab40854.

This ICC/IF data was generated using the same anti-Smad3 antibody clone, EP568Y, in a different buffer formulation (cat# ab40854).

ab40854 staining Smad3 in human granulosa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with ethanol and triton and blocked for 1 hour at 26°C. Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. An undiluted IRDye^® 800CW-conjugated goat anti-rabbit IgG (H+L) polyclonal was used as the secondary antibody. Left - negative control (4 replicates).

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