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

     

     

    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 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

  • 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 (IRDy 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.

  • 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.

     

  • 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.

  • 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.

     

  • 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.

  • 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.

  • 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.

  • 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.

  • 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

This product has been referenced in:
  • Gao L  et al. Dual inhibition of mTORC1/2 by DCZ0358 induces cytotoxicity in multiple myeloma and overcomes the protective effect of the bone marrow microenvironment. Cancer Lett 421:135-144 (2018). WB . Read more (PubMed: 29428642) »
  • Ding N  et al. miR-378a-3p exerts tumor suppressive function on the tumorigenesis of esophageal squamous cell carcinoma by targeting Rab10. Int J Mol Med 42:381-391 (2018). Read more (PubMed: 29693138) »
See all 13 Publications for this product

Customer reviews and Q&As

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1-2 of 2 Abreviews

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

Abcam user community

Verified customer

Submitted Jun 18 2018

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

Abcam user community

Verified customer

Submitted Aug 15 2016

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