Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Caspase-9 antibody [EPR18108] (ab185719)

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Overview

  • Product name

    Anti-Caspase-9 antibody [EPR18108]
    See all Caspase-9 primary antibodies

  • Description

    Rabbit monoclonal [EPR18108] to Caspase-9

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IPmore details

  • Species reactivity

    Reacts with: Mouse, Human

  • Immunogen

    Recombinant fragment within Human Caspase-9 aa 100-350. The exact sequence is proprietary.
    Database link: P55211

  • Positive control

    • WB: HeLa whole cell lysates untreated and treated with staurosporine 1uM for 4 hours; NIH/3T3 whole cell lysates untreated and treated with staurosporine1uM for 4 hours; Human fetal brain, fetal heart, fetal kidney and fetal liver and pancreas lysates; Mouse heart and kidney lysates. IP: HeLa whole cell lysate treated with staurosporine 1uM for 4 hours.

  • 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 ab185719 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. Detects a band of approximately 46, 37, 35 kDa (predicted molecular weight: 46 kDa).
IP 1/40.

Target

  • Function

    Involved in the activation cascade of caspases responsible for apoptosis execution. Binding of caspase-9 to Apaf-1 leads to activation of the protease which then cleaves and activates caspase-3. Proteolytically cleaves poly(ADP-ribose) polymerase (PARP).
    Isoform 2 lacks activity is an dominant-negative inhibitor of caspase-9.

  • Tissue specificity

    Ubiquitous, with highest expression in the heart, moderate expression in liver, skeletal muscle, and pancreas. Low levels in all other tissues. Within the heart, specifically expressed in myocytes.

  • Sequence similarities

    Belongs to the peptidase C14A family.
    Contains 1 CARD domain.

  • Developmental stage

    Expressed at low levels in fetal heart, at moderate levels in neonate heart, and at high levels in adult heart.

  • Post-translational
    modifications

    Cleavages at Asp-315 by granzyme B and at Asp-330 by caspase-3 generate the two active subunits. Caspase-8 and -10 can also be involved in these processing events.
  • Information by UniProt

  • Database links

    • Alternative names

      • APAF-3 antibody
      • APAF3 antibody
      • Apoptosis related cysteine peptidase antibody
      • Apoptotic protease Mch-6 antibody
      • Apoptotic protease-activating factor 3 antibody
      • CASP-9 antibody
      • CASP9 antibody
      • CASP9_HUMAN antibody
      • Caspase 9 apoptosis related cysteine peptidase antibody
      • Caspase 9 Dominant Negative antibody
      • Caspase 9c antibody
      • Caspase-9 antibody
      • Caspase-9 subunit p10 antibody
      • ICE LAP6 antibody
      • ICE like apoptotic protease 6 antibody
      • ICE-LAP6 antibody
      • ICE-like apoptotic protease 6 antibody
      • MCH6 antibody
      • PPP1R56 antibody
      • protein phosphatase 1, regulatory subunit 56 antibody
      • RNCASP9 antibody
      see all

    Images

    • Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)

      Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: Caspase-9 knockout HAP1 cell lysate (20 µg)
      Lane 3: HeLa cell lysate (20 µg)
      Lane 4: Jurkat cell lysate (20 µg)
      Lanes 1 - 4: Merged signal (red and green). Green - ab185719 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
      ab185719 was shown to specifically react with Caspase-9 when Caspase-9 knockout samples were used. Wild-type and Caspase-9 knockout samples were subjected to SDS-PAGE. ab185719 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10000 respectively 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 1/10000 dilution for 1 h at room temperature before imaging.

    • Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/10000 dilution

      Lane 1 : Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
      Lane 2 : HeLa whole cell lysate treated with staurosporine 1uM for 4 hours

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 46 kDa
      Observed band size: 35,37,46 kDa
      why is the actual band size different from the predicted?


      Exposure time: 10 seconds


      Blocking and dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/2500 dilution

      Lane 1 : Untreated NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate
      Lane 2 : NIH/3T3 whole cell lysate treated with staurosporine 1uM for 4 hours

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 46 kDa
      Observed band size: 37,39,49 kDa why is the actual band size different from the predicted?


      Exposure time: 30 seconds


      Blocking and dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/1000 dilution

      Lane 1 : Human fetal brain lysate
      Lane 2 : Human fetal heart lysate
      Lane 3 : Human fetal kidney lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

      Predicted band size: 46 kDa
      Observed band size: 46 kDa


      Exposure time: 30 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/5000 dilution

      Lane 1 : Human pancreas lysate
      Lane 2 : Human fetal liver lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

      Predicted band size: 46 kDa
      Observed band size: 46 kDa


      Exposure time: 30 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/1000 dilution

      Lane 1 : Mouse heart lysate
      Lane 2 : Mouse kidney lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

      Predicted band size: 46 kDa
      Observed band size: 37,39,49 kDa why is the actual band size different from the predicted?


      Exposure time: 15 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunoprecipitation - Anti-Caspase-9 antibody [EPR18108] (ab185719)
      Immunoprecipitation - Anti-Caspase-9 antibody [EPR18108] (ab185719)

      Caspase-9 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated with staurosporine 1uM for 4 hours with ab185719 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab185719 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

      Lane 1: HeLa whole cell lysate treated with staurosporine 10 µg (Input).

      Lane 2: ab185719 IP in HeLa whole cell lysate treated with staurosporine.

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab185719 in HeLa whole cell lysate treated with staurosporine.
      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    References

    This product has been referenced in:

    • Luff SA  et al. Role of p53 and transcription-independent p53-induced apoptosis in shear-stimulated megakaryocytic maturation, particle generation, and platelet biogenesis. PLoS One 13:e0203991 (2018). Read more (PubMed: 30231080) »
    See 1 Publication for this product

    Publishing research using ab185719? Please let us know so that we can cite the reference in this datasheet.

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