Anti-SMAD5 antibody [EP619Y] - BSA and Azide free

6 product images

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  Flow Cytometry (Intracellular) - Anti-SMAD5 antibody [EP619Y] - BSA and Azide free (ab238952)

  Overlay histogram showing PC-12 cells fixed in 4% PFA and stained with purified Anti-SMAD5 antibody [EP619Y] ab40771 at a dilution of 1/100 (red line). The secondary antibody used was Alexa Fluor^® 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).
  

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD5 antibody [EP619Y] ab40771).

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  Immunocytochemistry/ Immunofluorescence - Anti-SMAD5 antibody [EP619Y] - BSA and Azide free (ab238952)

  Immunofluorescence staining of HeLa cells with purified Anti-SMAD5 antibody [EP619Y] 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 Anti-SMAD5 antibody [EP619Y] 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.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD5 antibody [EP619Y] ab40771).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD5 antibody [EP619Y] - BSA and Azide free (ab238952)

  Unpurified Anti-SMAD5 antibody [EP619Y] 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.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD5 antibody [EP619Y] ab40771).

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  Flow Cytometry (Intracellular) - Anti-SMAD5 antibody [EP619Y] - BSA and Azide free (ab238952)

  Overlay histogram showing HEK293 cells stained with unpurified Anti-SMAD5 antibody [EP619Y] 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 (Anti-SMAD5 antibody [EP619Y] 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.
  

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD5 antibody [EP619Y] ab40771).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD5 antibody [EP619Y] - BSA and Azide free (ab238952)

  Immunohistochemical staining of paraffin embedded human testis with purified Anti-SMAD5 antibody [EP619Y] 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.
  

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD5 antibody [EP619Y] ab40771).

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  Western blot - Anti-SMAD5 antibody [EP619Y] - BSA and Azide free (ab238952)

  This data was developed using Anti-SMAD5 antibody [EP619Y] ab40771, the same antibody clone in a different buffer formulation.

  Western blot: Anti-SMAD3 (phospho S423 + S425) antibody [EP619Y] (Anti-SMAD5 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, Anti-SMAD5 antibody [EP619Y] 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] (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), ab255448 cell lysate at 20 µg

  Lane 8: Wild-type HeLa Treated TGF-beta (10 ng/mL, 30 min), 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), ab259776 cell lysate at 20 µg

  Lane 12: Wild-type HEK293 Treated TGF-beta (10 ng/mL, 30 min), 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 - 14: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 14: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 52 kDa