Anti-Importin4 antibody (ab28387)

Anti-Importin4 antibody images

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  Western blot - Anti-Importin4 antibody (ab28387)This image is courtesy of the laboratory of Genevieve Almouzni
  All lanes : Anti-Importin4 antibody (ab28387) at 1 µg/ml
  
  Lane 1 : Hela B Cell Lysate at 30 µg
  Lane 2 : Hela B Cell Lysate at 60 µg
  
  
  Predicted band size : 119 kDa
  Observed band size : 130 kDa (why is the actual band size different from the predicted?)
  Additional bands at : 170 kDa (possible glycosylated form,non-specific binding).

  This image is courtesy of the laboratory of Genevieve Almouzni

  Importin4 antibody ab28387 detected a 130 kDa doublet in Hela B lysate that is likely to correspond to Importin4. Staining of tubulin in the same lanes is shown as a loading control. The antibody was also able to detect recombinant Importin4 protein but not recombinant Importin5 protein (not shown).
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  Western blot - Importin4 antibody (ab28387)
  All lanes : Anti-Importin4 antibody (ab28387) at 1 µg/ml
  
  Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
  Lane 2 : HeLa nuclear extract lysate (TPA stimulated 2 hr) (ab14653)
  
  Lysates/proteins at 20 µg per lane.
  
  Secondary
  Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
  
  Performed under reducing conditions.
  
  Predicted band size : 119 kDa
  Observed band size : 119 kDa
  Additional bands at : 116 kDa. We are unsure as to the identity of these extra bands.
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  Immunocytochemistry/ Immunofluorescence - Anti-Importin4 antibody (ab28387)
  ICC/IF image of ab28387 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab28387, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
  
  
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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Importin4 antibody (ab28387)
  IHC image of ab28387 staining in human skin formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab28387, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.