Anti-Smad3 antibody [EP568Y]

19 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

  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.

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  Flow Cytometry (Intracellular) - Anti-Smad3 antibody [EP568Y] (ab40854)

  Intracellular 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.
  

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  This image is courtesy of a customer review submitted by Francesco Elia Marino

  Immunocytochemistry/ Immunofluorescence - Anti-Smad3 antibody [EP568Y] (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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  Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

  Blocking buffer and concentration: 5% NFDM/TBST.

  Diluting buffer and concentration: 5% NFDM /TBST.

  All lanes: Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5300 dilution

  Lane 1: HT-29 cell lysate at 10 µg

  Lane 2: HT-1080 cell lysate at 10 µg

  Secondary

  All lanes: Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

  Predicted band size: 48 kDa

  Observed band size: 58 kDa

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  Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

  All lanes: Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5000 dilution

  All lanes: Jurkat cell lysate at 10 µg

  Predicted band size: 48 kDa

  Observed band size: 55 kDa

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  Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

  Lanes 1 - 4: Merged signal (red and green). Green - ab40854 observed at 48 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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: Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/1000 dilution

  Lane 1: Wild-type A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

  Lane 2: Western blot - Human SMAD3 knockout A549 cell lysate (Human SMAD3 knockout A549 cell lysate ab264513)

  Lane 3: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

  Lane 4: Human Kidney cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 48 kDa

  Observed band size: 50 kDa

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  Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

  Blocking buffer and concentration: 5% NFDM/TBST.

  Diluting buffer and concentration: 5% NFDM /TBST.

  All lanes: Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5300 dilution

  All lanes: Rat liver tissue lysate at 10 µg

  Secondary

  All lanes: Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

  Predicted band size: 48 kDa

  Observed band size: 58 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Smad3 antibody [EP568Y] (ab40854)

  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 (green, left panel). Counterstained with DAPI (blue, right panel).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

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

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

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

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

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

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

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

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Flow Cytometry (Intracellular) - Anti-Smad3 antibody [EP568Y] (ab40854)

  Overlay histogram showing HCT116 cells stained with unpurifiedab40854 (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 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
  

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  Sandwich ELISA - Anti-Smad3 antibody [EP568Y] (ab40854)

  Standard Curve for Smad3 (Analyte: Smad3 protein (His tag) (Recombinant Human Smad3 protein 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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

  Immunohistochemical analysis of paraffin-embedded human colonic adenocarcinoma tissue labelling Smad3 with unpurified ab40854.
  

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  ChIC/CUT&RUN sequencing - Anti-Smad3 antibody [EP568Y] (ab40854)

  ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab40854 [EP568Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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  Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

  False colour image of Western blot: Anti-Smad3 antibody [EP568Y] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40854 was shown to bind specifically to Smad3. A band was observed at 50 kDa in wild-type A549 cell lysates with no signal observed at this size in SMAD3 CRISPR-Cas9 edited cell line Human SMAD3 knockout A549 cell line ab277888 (CRISPR-Cas9 edited cell lysate None). The band observed in the CRISPR-Cas9 edited lysate lane below 50 kDa is likely to represent a truncated form of Smad3. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and SMAD3 CRISPR-Cas9 edited A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

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

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: SMAD3 CRISPR-Cas9 edited A549 cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 2: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 48 kDa

  Observed band size: 50 kDa

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  ChIC/CUT&RUN sequencing - Anti-Smad3 antibody [EP568Y] (ab40854)

  ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab40854 [EP568Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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  ChIC/CUT&RUN sequencing - Anti-Smad3 antibody [EP568Y] (ab40854)

  ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab40854 [EP568Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.