Validated using a knockout cell line
Recombinant
RabMAb

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

Reviews (1)Q&A (1)References (7)

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

     

     

    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.

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. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Antigen retrieval is recommended.
  • Application notes
    Is unsuitable for Flow Cyt or IP.

    • Target

    Images

    • Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)

      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.

    • Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)

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

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)

      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.

    • Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      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

    • Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      Western blot - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      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

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Pro Caspase-8 antibody [EPR162] (ab108333)

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

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    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) »
    • Wang Q  et al. Efficacy of celastrol combined with cisplatin in enhancing the apoptosis of U-2OS osteosarcoma cells via the mitochondrial and endoplasmic reticulum pathways of apoptosis. Oncol Lett 17:3305-3313 (2019). Read more (PubMed: 30867764) »
    See all 7 Publications for this product

    Publishing research using ab108333? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-2 of 2 Abreviews or Q&A

    Question

    I have purchased a product from your company for my research work, I would like to know if the the product antibody can also detect the cleaved caspase -8 subunits,larger subunit p18 and smaller subunit p10, along with 55kDa . As it is imp for me to detect cleaved caspase 8 subunits for my research work. I have also read the product datasheet and it has confused me as it has not been mentioned clearly. The product datasheet reads , Specificity-A synthetic peptide corresponding to residues in human Caspase-8 was used as an immunogen. Please help me out regarding it. Product details caspase-8 rabbit monoclonal antibody clone ID: EPR162.

    Read More
    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

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Caspase 8 antibody [EPR162]

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

    Dr. Kirk Mcmanus

    Verified customer

    Submitted Aug 09 2012

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