Anti-SMAD5 antibody [EP619Y]

9 product images

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  Flow Cytometry (Intracellular) - Anti-SMAD5 antibody [EP619Y] (ab40771)

  Overlay histogram showing PC-12 cells fixed in 4% PFA and stained with purified ab40771 at a dilution of 1/100 (red line). The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit at a dilution of 1/500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).
  

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  Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771)

  Blocking and dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/1000 dilution

  Lane 1: 3T3-L1 (Mouse embryonic fibroblast) lysate at 20 µg

  Lane 2: Neuro-2a (Mouse neuroblastoma neuroblast) at 20 µg

  Lane 3: F9 (Mouse embryonal carcinoma epithelial cell) lysate at 20 µg

  Secondary

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

  Predicted band size: 52 kDa

  Observed band size: 52 kDa

  Exposure time: 3min

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  Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771)

  Blocking buffer: 5% NFDM/TBST
  Dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/5000 dilution

  Lane 1: HEK293 whole cell lysate at 10 µg

  Lane 2: COS-1 whole cell lysate at 10 µg

  Secondary

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

  Predicted band size: 52 kDa

  Observed band size: 52 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-SMAD5 antibody [EP619Y] (ab40771)

  Immunofluorescence staining of HeLa cells with purified ab40771 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077), used at a dilution of 1/1000. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab40771 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at a dilution of 1/500. For negative control 2, Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at a dilution of 1/400.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD5 antibody [EP619Y] (ab40771)

  Unpurified ab40771 (4μg/ml) staining SMAD5 in human skin using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of nuclear/cytoplasmic compartments within the stratum granulosum.
  Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD5 antibody [EP619Y] (ab40771)

  Immunohistochemical staining of paraffin embedded human testis with purified ab40771 at a working dilution of 1/50. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

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  Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771)

  All lanes: Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/1000 dilution

  All lanes: Cos-1 cell lysate at 10 µg

  Predicted band size: 52 kDa

  Observed band size: 52 kDa

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  Flow Cytometry (Intracellular) - Anti-SMAD5 antibody [EP619Y] (ab40771)

  Overlay histogram showing HEK293 cells stained with unpurified ab40771 (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 (ab40771, 1/50 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 monoclonal IgG (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
  

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  Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771)

  Western blot: Anti-SMAD3 (phospho S423 + S425) antibody [EP619Y] (ab40771) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab40771 was shown to bind specifically to SMAD3 (phospho S423 + S425). A band was observed at 52 kDa in wild-type HeLa cell lysates with no signal observed at this size in SMAD3 (phospho S423 + S425) knockout cell line. To generate this image, wild-type and SMAD3 (phospho S423 + S425) knockout HeLa 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-SMAD5 antibody [EP619Y] (ab40771) at 1/1000 dilution

  Lane 1: Wild-type HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min) cell lysate at 20 µg

  Lane 2: Wild-type HeLa Treated TGF-beta (10 ng/mL, 30 min) cell lysate at 20 µg

  Lane 3: SMAD1 knockout HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min), Human SMAD1 knockout HeLa cell line ab265400 cell lysate at 20 µg

  Lane 4: SMAD1 knockout HeLa Treated TGF-beta (10 ng/mL, 30 min), Human SMAD1 knockout HeLa cell line ab265400 cell lysate at 20 µg

  Lane 5: SMAD2 knockout HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min), Human SMAD2 knockout HeLa cell line ab255430 cell lysate at 20 µg

  Lane 6: SMAD2 knockout HeLa Treated TGF-beta (10 ng/mL, 30 min), Human SMAD2 knockout HeLa cell line ab255430 cell lysate at 20 µg

  Lane 7: Wild-type HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min), Human wild-type HeLa cell line ab255448 cell lysate at 20 µg

  Lane 8: Wild-type HeLa Treated TGF-beta (10 ng/mL, 30 min), Human wild-type HeLa cell line ab255448 cell lysate at 20 µg

  Lane 9: SMAD3 knockout HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min), Human SMAD3 knockout HeLa cell line ab255431 cell lysate at 20 µg

  Lane 10: SMAD3 knockout HeLa Treated TGF-beta (10 ng/mL, 30 min), Human SMAD3 knockout HeLa cell line ab255431 cell lysate at 20 µg

  Lane 11: Wild-type HEK293 Vehicle Control TGF-beta (0 ng/mL, 30 min), Human wild-type HEK-293 cell line ab259776 cell lysate at 20 µg

  Lane 12: Wild-type HEK293 Treated TGF-beta (10 ng/mL, 30 min), Human wild-type HEK-293 cell line ab259776 cell lysate at 20 µg

  Lane 13: SMAD5 knockout HEK293 Vehicle Control TGF-beta (0 ng/mL, 30 min), Human SMAD5 knockout HEK-293 cell line ab269470 cell lysate at 20 µg

  Lane 14: SMAD5 knockout HEK293 Treated TGF-beta (10 ng/mL, 30 min), Human SMAD5 knockout HEK-293 cell line ab269470 cell lysate at 20 µg

  Secondary

  Lanes 1, 10, 11, 12, 13, 14, 2, 3, 4, 5, 6, 7, 8 and 9: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

  Lanes 1, 10, 11, 12, 13, 14, 2, 3, 4, 5, 6, 7, 8 and 9: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 52 kDa