Anti-SMAD5 antibody [EP619Y] (ab40771)

Anti-SMAD5 antibody [EP619Y] images

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  Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771)
  Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/5000 dilution (purified) + PC-12 whole cell lysate at 10 µg
  
  Secondary
  Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (HRP goat anti-rabbit IgG (H+L))
  
  Predicted band size : 52 kDa
  

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

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  Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771)
  All lanes : Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/5000 dilution (purified)
  
  Lane 1 : HEK293 whole cell lysate
  Lane 2 : COS-1 whole cell lysate
  
  Lysates/proteins at 10 µg per lane.
  
  Secondary
  Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (HRP goat anti-rabbit IgG (H+L))
  
  Predicted band size : 52 kDa
  Observed band size : 52 kDa
  

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

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  Flow Cytometry - 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 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).

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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 (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with 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 (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

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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 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 - SMAD5 antibody [EP619Y] (ab40771)
  Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/1000 dilution (unpurified) + Cos-1 cell lysate at 10 µg
  
  Predicted band size : 52 kDa
  Observed band size : 52 kDa
  
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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)-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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  Flow Cytometry-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) (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.