Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Pro Caspase-8 antibody [EPR162] (ab108333)

Overview

  • Product name
    Anti-Pro Caspase-8 antibody [EPR162]
    See all Pro Caspase-8 primary antibodies
  • Description
    Rabbit monoclonal [EPR162] to Pro Caspase-8
  • Host species
    Rabbit
  • Specificity

    Theoretically, ab108333 should recognize p55/54, p43/41 and p18. However, in our internal tests, it only recognized pro caspase 8. We tested it side by side with ab32397. Both antibodies are KO validated, and ab32397 recognizes pro-caspase 8 and cleavage caspase 8.

  • Tested applications
    Suitable for: WB, IHC-Pmore details
    Unsuitable for: Flow Cyt or IP
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Pro Caspase-8 aa 350-450 (internal sequence). The exact sequence is proprietary.
    Database link: Q14790

  • Positive control
    • Jurkat, IM9, HepG2, HeLa, and HL60 cell lysates; Human tonsil tissue and Human hepatocellular carcinoma tissue; HeLa cells.
  • General notes

     

     

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab108333 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000 - 1/10000. Detects a band of approximately 55 kDa (predicted molecular weight: 55 kDa).
IHC-P 1/100 - 1/250. Antigen retrieval is recommended.
  • Application notes
    Is unsuitable for Flow Cyt or IP.
  • Target

    Images

    • Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: Pro Caspase-8 knockout HAP1 cell lysate (20 µg)
      Lanes 1 and 2: Merged signal (red and green). Green - ab108333 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab108333 was shown to specifically react with Caspase-8 when Caspase-8 knockout samples were used. Wild-type and Pro Caspase-8 knockout samples were subjected to SDS-PAGE. ab108333 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

    • All lanes : Anti-Pro Caspase-8 antibody [EPR162] (ab108333) at 1/1000 dilution

      Lane 1 : HepG2 whole cell lysate
      Lane 2 : Rat liver tissue lysate
      Lane 3 : Mouse liver tissue lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

      Performed under reducing conditions.

      Predicted band size: 55 kDa
      Observed band size: 55 kDa



      Lanes 1 - 3: Merged signal (red and green). Green - ab108333 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa. 

      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 using Licor blocking buffer before being incubated with ab108333 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

    • ab108333, at 1/100 dilution, staining Pro Caspase-8 in paraffin-embedded human tonsil tissue by Immunohistochemistry.

    • Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: Pro Caspase-8 knockout HAP1 cell lysate (20 µg)
      Lanes 1 and 2: Merged signal (red and green).

      Green - target observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

      This western blot image is a comparison between ab108333 and a competitor's top cited mouse monoclonal antibody.

    • All lanes : Anti-Pro Caspase-8 antibody [EPR162] (ab108333) at 1/1000 dilution

      Lane 1 : Jurkat cell lysate
      Lane 2 : IM9 cell lysate
      Lane 3 : HepG2 cell lysate
      Lane 4 : HL60 cell lysate

      Lysates/proteins at 10 µg per lane.

      Predicted band size: 55 kDa

    • All lanes : Anti-Pro Caspase-8 antibody [EPR162] (ab108333) at 1/1000 dilution

      Lane 1 : Jurkat cell lysate, treated with etoposide
      Lane 2 : Jurkat cell lysate
      Lane 3 : HeLa cell lysate, treated staurosporine
      Lane 4 : HeLa cell lysate

      Lysates/proteins at 10 µg per lane.

      Predicted band size: 55 kDa

    • ab108333, at 1/100 dilution, staining Pro Caspase-8 in paraffin-embedded human hepatocellular carcinoma by Immunohistochemistry.

    References

    This product has been referenced in:
    • Liao Y  et al. Endoplasmic Reticulum Stress Induces Macrophages to Produce IL-1ß During Mycobacterium bovis Infection via a Positive Feedback Loop Between Mitochondrial Damage and Inflammasome Activation. Front Immunol 10:268 (2019). Read more (PubMed: 30846986) »
    • Pittala S  et al. Targeting Liver Cancer and Associated Pathologies in Mice with a Mitochondrial VDAC1-Based Peptide. Neoplasia 20:594-609 (2018). WB ; Mouse . Read more (PubMed: 29747160) »
    See all 4 Publications for this product

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Answer


    The immunogen sequence is considered proprietary information. The sequence is a 30-amino acid sequence within the larger range of amino acids from 200 - 250 of the human protein sequence that is given in the UniProt entry at this link: http://www.uniprot.org/uniprot/Q14790.

    Part of the sequence is in the p18 subunit and part of it is in the N-terminus propeptide. The western blot data on the datasheet suggests that the antibody is not capable of detecting the p18 subunit, or the smaller p10 subunit.

    By comparison, the mouse monoclonal antibody ab119809 (clone M382) appears to be capable of detecting the full-length inactive caspase 8 and also the p10 subunit. However, we do not know of any published data for this clone.

    Read More
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Specification
    HeLa
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.5% Triton-X100 in PBS

    Dr. Kirk Mcmanus

    Verified customer

    Submitted Aug 09 2012

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