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Anti-Islet 1 antibody [1H9] (ab86472)

Key features and details

  • Mouse monoclonal [1H9] to Islet 1
  • Suitable for: Flow Cyt, WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Human, Recombinant fragment
  • Isotype: IgG1

Overview

  • Product name

    Anti-Islet 1 antibody [1H9]
    See all Islet 1 primary antibodies

  • Description

    Mouse monoclonal [1H9] to Islet 1

  • Host species

    Mouse

  • Specificity

    In western blot, we observe a specific band at ~40kDa which is not seen in KO cell lines. A cross-reactive band was observed in the wild-type and knockout cells. The additional band below this band of interest is seen at ~38kDa in both the WT and KO cells. We are unsure as to the identity of these extra bands.

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    WB
    Human
    Recombinant fragment
    See all applications and species data

  • Immunogen

    Recombinant fragment corresponding to Human Islet 1 aa 150-349. Purified recombinant fragment of Human Islet 1 expressed in E. Coli
    Database link: P61371

  • Positive control

    • Transfected HEK293 cell lysate. Human lung cancer tissue and cervical carcinoma tissue.

  • General notes

    This product was changed from ascites to supernatant. Lot no’s high than GR141006-29 are from Tissue Culture Supernatant

Properties

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Islet 1 antibody [1H9] (ab86472)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Islet 1 antibody [1H9] (ab86472)
    ab86472 at a 1/200 dilution staining Islet 1 in human lung cancer tissue. DAB staining shows nuclear localisation.
  • Western blot - Anti-Islet 1 antibody [1H9] (ab86472)
    Western blot - Anti-Islet 1 antibody [1H9] (ab86472)
    All lanes : Anti-Islet 1 antibody [1H9] (ab86472) at 1/500 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : ISL1 (Islet 1) knockout HAP1 whole cell lysate
    Lane 3 : SH-SY5Y whole cell lysate
    Lane 4 : HepG2 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 39 kDa
    Observed band size: 40 kDa
    why is the actual band size different from the predicted?



    Lanes 1 - 4: Merged signal (red and green). Green - ab86472 observed at 40 kDa. Red - loading control, ab176560, observed at 50 kDa.

    ab86472 was shown to recognize Islet 1 in wild-type HAP1 cells as signal was lost at the expected MW in Islet 1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Islet 1 knockout samples were subjected to SDS-PAGE. ab86472 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Western blot - Anti-Islet 1 antibody [1H9] (ab86472)
    Western blot - Anti-Islet 1 antibody [1H9] (ab86472)
    Anti-Islet 1 antibody [1H9] (ab86472) at 1/500 dilution + HEK293 cells transfected with full length Islet 1 (aa1-349)-hIgGFc

    Predicted band size: 39 kDa



    Note: The molecular weight is larger than expected, as the recombinant protein is not the endogenouse protein, but rather the fc-fusion protein

  • Immunocytochemistry/ Immunofluorescence - Anti-Islet 1 antibody [1H9] (ab86472)
    Immunocytochemistry/ Immunofluorescence - Anti-Islet 1 antibody [1H9] (ab86472)

    Confocal immunofluorescence analysis of HEK293 cells trasfected with full-length Islet 1-hIgGFc using ab86472 at 1/150 dilution (green). Blue: DRAQ5 fluorescent DNA dye.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Islet 1 antibody [1H9] (ab86472)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Islet 1 antibody [1H9] (ab86472)
    ab86472 at a 1/200 dilution staining Islet 1 in human cervical carcinoma tissue. DAB staining shows nuclear localization.
  • Flow Cytometry - Anti-Islet 1 antibody [1H9] (ab86472)
    Flow Cytometry - Anti-Islet 1 antibody [1H9] (ab86472)

    Overlay histogram showing SH-SY5Y cells stained with ab86472 (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 (ab86472, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10^6 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive result in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween for 20 min used under the same conditions.

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