Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Caspase-9 antibody [EPR18107] (ab202068)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR18107] to Caspase-9
  • Suitable for: IHC-P, WB, ICC/IF, IP
  • Knockout validated
  • Reacts with: Mouse, Human

Overview

  • Product name

    Anti-Caspase-9 antibody [EPR18107]
    See all Caspase-9 primary antibodies

  • Description

    Rabbit monoclonal [EPR18107] to Caspase-9

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IHC-P, WB, ICC/IF, IPmore details

  • Species reactivity

    Reacts with: Mouse, Human

  • Immunogen

    Recombinant fragment within Human Caspase-9 aa 100-300. The exact sequence is proprietary.
    Database link: P55211

  • Positive control

    • WB: HeLa and C2C12 whole cell lysates; Human fetal brain, fetal heart, fetal kidney and fetal liver lysates. IHC-P: Human cervix carcinoma tissue. ICC/IF: HeLa cells. IP: HeLa treated with staurosporine 1uM for 4 hours whole cell lysate.

  • 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 ab202068 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
IHC-P 1/300. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/2000. Detects a band of approximately 46, 35, 37 kDa (predicted molecular weight: 46 kDa).
ICC/IF 1/500.
IP 1/80.

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 [EPR18107] (ab202068)
      Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)

      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 - ab202068 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
      ab202068 was shown to recognize Caspase-9 when Caspase-9 knockout samples were used, along with additional cross-reactive bands. Wild-type and Caspase-9 knockout samples were subjected to SDS-PAGE. ab202068 and ab8245 (loading control to GAPDH) were diluted at 1/2000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed withGoat 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.

    • Immunocytochemistry/ Immunofluorescence - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      Immunocytochemistry/ Immunofluorescence - Anti-Caspase-9 antibody [EPR18107] (ab202068)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Caspase-9 with ab202068 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

      Confocal image showing cytoplasmic and nuclear staining on HeLa cell line. The expression increased after treatment with staurosporine (1uM) for 4 hours.

      The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

      The negative controls are as follows:
      -ve control 1: ab202067 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
      -ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [EPR18107] (ab202068)

      Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling Caspase-9 with ab202068 at 1/300 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

      Cytoplasmic and nuclear staining on Human cervix carcinoma tissue is observed.

      Counter stained with Hematoxylin.

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

      Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    • Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)

      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 - target observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.

      This western blot image is a comparison between ab202068 and a competitor's top cited rabbit polyclonal antibody.

       

    • Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/10000 dilution

      Lane 1 : Untreated C2C12 (Mouse myoblast cell line) whole cell lysate
      Lane 2 : C2C12 (Mouse myoblast cell line) treated with staurosporine 1uM for 4 hours whole cell 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: 37,39,46 kDa
      why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/2000 dilution

      Lane 1 : Human fetal brain lysate
      Lane 2 : Human fetal heart 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: 46 kDa


      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/2000 dilution

      Lane 1 : Human fetal kidney lysate
      Lane 2 : Human fetal liver 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: 46 kDa


      Exposure time: 30 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunoprecipitation - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      Immunoprecipitation - Anti-Caspase-9 antibody [EPR18107] (ab202068)

      Caspase-9 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) treated with staurosporine 1uM for 4 hours whole cell lysate with ab202068 at 1/80 dilution.

      Western blot was performed from the immunoprecipitate using ab202068 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 treated with staurosporine 1uM for 4 hours whole cell lysate10 µg (Input).

      Lane 2: ab202068 IP in HeLa treated with staurosporine 1uM for 4 hours whole cell lysate.

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202068 in HeLa treated with staurosporine 1uM for 4 hours whole cell lysate.
      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 3 seconds.

    • Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)
      All lanes : Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/50000 dilution

      Lane 1 : Untreated HeLa (human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg
      Lane 2 : HeLa (human epithelial cells from cervix adenocarcinoma) treated with staurosporine 1 µM for 4 hours whole cell lysate at 20 µg

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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



      Blocking/Dilution buffer: 5% NFDM/TBST.

    References

    Publishing research using ab202068? Please let us know so that we can cite the reference in this datasheet.

    ab202068 has been referenced in 43 publications.

    • Liu D  et al. Protective Effects of Chlorogenic Acid on Cerebral Ischemia/Reperfusion Injury Rats by Regulating Oxidative Stress-Related Nrf2 Pathway. Drug Des Devel Ther 14:51-60 (2020). PubMed: 32021091
    • Ren Z  et al. Effect of intranasal instillation of Escherichia coli on apoptosis of spleen cells in diet-induced-obese mice. Sci Rep 10:5109 (2020). PubMed: 32198370
    • Fu G  et al. CDCA5 functions as a tumor promoter in bladder cancer by dysregulating mitochondria-mediated apoptosis, cell cycle regulation and PI3k/AKT/mTOR pathway activation. J Cancer 11:2408-2420 (2020). PubMed: 32201512
    • Liu Y  et al. PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4+ and CD8+ T Cells During BVDV Infection in vitro. Front Immunol 11:467 (2020). PubMed: 32256500
    • Wang L  et al. ADAM12 silencing promotes cellular apoptosis by activating autophagy in choriocarcinoma cells. Int J Oncol 56:1162-1174 (2020). PubMed: 32319603
    View all Publications for this product

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    1-2 of 2 Abreviews

    Western blot abreview for Anti-Caspase-9 antibody [EPR18107]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (B16 cells)
    Gel Running Conditions
    Reduced Denaturing (4-20% Tris Glycin gel, semi dry transfer)
    Loading amount
    50 µg
    Specification
    B16 cells
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jun 18 2018

    Western blot abreview for Anti-Caspase-9 antibody [EPR18107]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (OV90 cells)
    Gel Running Conditions
    Reduced Denaturing (10% Tris Glycin gel)
    Loading amount
    50000 cells
    Specification
    OV90 cells
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More

    Abcam user community

    Verified customer

    Submitted Aug 15 2016

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