Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E235] to EGFR - BSA and Azide free
  • Suitable for: WB, IP, Flow Cyt, ICC/IF, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-EGFR antibody [E235] - BSA and Azide free
    See all EGFR primary antibodies

  • Description

    Rabbit monoclonal [E235] to EGFR - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IP, Flow Cyt, ICC/IF, IHC-Pmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: A431, HeLa and MDA-MB-468 cell lysate. ICC/IF: A431 cells. Flow Cyt: A431 cells. IHC-P: FFPE mouse skin normal. IP: A431 cell lysate

  • General notes

    Ab227459 is the carrier-free version of ab32077. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with

    ab227459 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

Images

  • Western blot - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
    Western blot - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
    All lanes : Anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution

    Lane 1 : A431 cell lysate
    Lane 2 : MDA-MB-468 cell lysate
    Lane 3 : Wild type HeLa cell lysate
    Lane 4 : EGFR knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 134 kDa
    Observed band size: 134 kDa



    This data was developed using the same antibody clone in a different buffer formulation (ab32077).

    Lanes 1 - 4: Merged signal (red and green). Green - ab32077 observed at 175 kDa. Red - loading control, ab130007 observed at 125 kDa.  

     ab32077 was shown to react with EGFR in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255385 (knockout cell lysate ab263845) was used. Wild-type and EGFR knockout samples were subjected to SDS-PAGE. ab32077 and Anti-Vinculin antibody [VIN-54] (ab130007) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

     

  • Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
    Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)

    Immunocytochemistry/Immunofluorescence analysis of A431 cells labelling EGFR with ab32077 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
    Control: PBS only.
    Nuclear counter stain: DAPI.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32077).

  • Flow Cytometry - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
    Flow Cytometry - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)

    Overlay histogram showing A431 cells stained with ab32077 (red line). The cells were fixed with 80% 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 (ab32077, 1/1000 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 IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32077).

  • Immunoprecipitation - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
    Immunoprecipitation - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)

    Purified ab32077 at 1/50 dilution (2µg) immunoprecipitating EGFR in A431 whole cell lysate.
    Lane 1 (input): A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10µg
    Lane 2 (+): ab32077 + A431 whole cell lysate.
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32077 in A431 whole cell lysate.
    VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
    Blocking Buffer and concentration: 5% NFDM/TBST.
    Diluting buffer and concentration: 5% NFDM/TBST.
    Observed band size: 180 kDa
    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32077).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)

    This IHC data was generated using the same anti-EGFR antibody clone, E235, in a different buffer formulation (cat# ab32077).

    IHC image of EGFR staining in a formalin fixed, paraffin embedded mouse normal skin 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 ab32077 at 1/200 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.

  • Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
    Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)

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