Anti-Cofilin (phospho S3) 抗体 (ab100836)

Anti-Cofilin (phospho S3) antibody 画像

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  Immunocytochemistry/ Immunofluorescence - Anti-Cofilin (phospho S3) antibody (ab100836)

  ICC/IF image of ab100836 stained MCF7 cells. The cells were 4% formaldehyde 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 (ab100836, 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. This antibody also gave a positive result in 4% formaldehyde fixed (10 min) HeLa cells at 5µg/ml,.

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  Western blot - Anti-Cofilin (phospho S3) antibody (ab100836)
  All lanes : Anti-Cofilin (phospho S3) antibody (ab100836) at 1 µg/ml
  
  Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
  Lane 2 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
  Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/ml
  Lane 4 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/ml
  Lane 5 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate with Non modified peptide at 1 µg/ml
  Lane 6 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate with Non modified peptide at 1 µg/ml
  
  Lysates/proteins at 15 µg per lane.
  
  Secondary
  Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
  Developed using the ECL technique
  
  Performed under reducing conditions.
  
  Predicted band size : 18 kDa
  Observed band size : 20 kDa (why is the actual band size different from the predicted?)
  Additional bands at : 110 kDa,150 kDa,33 kDa. We are unsure as to the identity of these extra bands.
  
  Exposure time : 180 seconds
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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cofilin (phospho S3) antibody (ab100836)
  IHC image of ab100836 staining in human breast adenocarcinoma 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 ab100836, 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.
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  ELISA - Anti-Cofilin (phospho S3) antibody (ab100836)

  ab100836 was tested using an Indirect ELISA approach. The wells were coated with peptide (1µg/ml at 100µl/well) overnight at 4°C, followed by a 5% BSA blocking step for 1 hour at room temperature. The primary Ab was then added at a dilution range of 1- 0.00025µg/ml (100µl/well) for 1hr at room temperature. A HRP-conjugated anti-rabbit IgG (heavy and light chain) was used as a secondary antibody at 1:20,000 dilution for 1hr at room temperature.