Anti-EGFR antibody [E114]

10 product images

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  Western blot - Anti-EGFR antibody [E114] (ab32562)

  This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab32562 overnight at 4°C in the presence of loading control ab18058 Mouse monoclonal [SPM227] to Vinculin diluted 1:10000). Antibody binding was detected using IR-labelled goat anti-Rabbit Ab at a 1:10,000 dilution for one hour at room temperature before imaging.
  

  This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR.

  All lanes: Western blot - Anti-EGFR antibody [E114] (ab32562) at 1/10000 dilution

  All lanes: A431 at 20 µg

  Performed under reducing conditions.

  Predicted band size: 134 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E114] (ab32562)

  IHC image of EGFR staining in mouse skin formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. 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 ab32562, 1in50 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC 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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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E114] (ab32562)

  IHC image of EGFR staining in rat skin formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab32562, 1in50 dilution, 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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  Immunoprecipitation - Anti-EGFR antibody [E114] (ab32562)

  ab32562 (purified) at 1/20 immunoprecipitating EGFR in HeLa whole cell lysate.
  

  Lane 1 (input): HeLa whole cell lysate (10μg)

  Lane 2 (+): ab32562 + HeLa whole cell lysate.

  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32562 in HeLa whole cell lysate.

  For western blotting, VeriBlot for IP Detection Reagent (HRP) ab131366 VeriBlot for IP (HRP) was used for detection (1/10000).

  Blocking buffer and concentration: 5% NFDM/TBST.

  Diluting buffer and concentration: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-EGFR antibody [E114] (ab32562)

  Predicted band size: 134 kDa

  Observed band size: 170 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E114] (ab32562)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling EGFR with purified ab32562 at a dilution of 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

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  Western blot - Anti-EGFR antibody [E114] (ab32562)

  Blocking and dilution buffer: 5% NFDM /TBST.

  All lanes: Western blot - Anti-EGFR antibody [E114] (ab32562) at 1/10000 dilution

  All lanes: A431 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 1/100000 dilution

  Predicted band size: 134 kDa

  Observed band size: 170 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E114] (ab32562)

  Immunocytochemistry/Immunofluorescence analysis of A431 cells labelling EGFR with purified ab32562 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

  Control 1: primary antibody (1/100) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

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  Western blot - Anti-EGFR antibody [E114] (ab32562)

  Blocking and dilution buffer: 5% NFDM /TBST.

  All lanes: Western blot - Anti-EGFR antibody [E114] (ab32562) at 1/10000 dilution

  All lanes: HeLa 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 1/100000 dilution

  Predicted band size: 134 kDa

  Observed band size: 170 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E114] (ab32562)

  IHC image of EGFR staining in human nomal cervix formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 ab32562, 1in50 dilution, 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.

  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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  Flow Cytometry (Intracellular) - Anti-EGFR antibody [E114] (ab32562)

  ab32562 staining EGFRin the human cell line A431 (human epidermoid carcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.
  

  Isoytype control: Rabbit monoclonal IgG (Black)

  Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)