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Cell Biology Apoptosis Intracellular Caspases etc Caspases
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Caspase-9 antibody [E23] (ab32539)

  • Datasheet
  • SDS
Reviews (5)Q&A (10)References (143)

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Western blot - Anti-Caspase-9 antibody [E23] (ab32539)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [E23] (ab32539)
  • Immunocytochemistry/ Immunofluorescence - Anti-Caspase-9 antibody [E23] (ab32539)
  • Flow Cytometry - Anti-Caspase-9 antibody [E23] (ab32539)
  • Immunoprecipitation - Anti-Caspase-9 antibody [E23] (ab32539)
  • Western blot - Anti-Caspase-9 antibody [E23] (ab32539)
  • Flow Cytometry - Anti-Caspase-9 antibody [E23] (ab32539)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [E23] (ab32539)
  • Western blot - Anti-Caspase-9 antibody [E23] (ab32539)
  • Anti-Caspase-9 antibody [E23] (ab32539)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E23] to Caspase-9
  • Suitable for: WB, IHC-P, Flow Cyt, IP, ICC/IF
  • Knockout validated
  • Reacts with: Human

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Overview

  • Product name

    Anti-Caspase-9 antibody [E23]
    See all Caspase-9 primary antibodies
  • Description

    Rabbit monoclonal [E23] to Caspase-9
  • Host species

    Rabbit
  • Specificity

    This antibody should recognise both the pro-[40kDa] form and p35 cleaved form of Caspase-9.
  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt, IP, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human Caspase-9 aa 250-350. The exact sequence is proprietary.
    Database link: P55211

  • Positive control

    • WB: Jurkat and HeLa whole cell lysate (ab150035). IHC-P: Human skeletal muscle and cervical carcinoma tissues. ICC/IF: HepG2 cells. Flow Cyt: K562 cells. IP: HeLa whole cell lysate (ab150035).
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    E23
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Caspases etc
    • Caspases
    • Cancer
    • Invasion/microenvironment
    • Apoptosis
    • Caspases
    • Cancer
    • Invasion/microenvironment
    • Apoptosis
    • Cytochrome C
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Apoptosis
    • Cancer
    • Cell Death
    • Apoptosis
    • Apoptosis Markers
    • Caspases
    • Cancer
    • Cell Death
    • Apoptosis
    • Apoptosis Markers
    • Cytochrome C
    • Cancer
    • Cell Death
    • Apoptosis
    • Metabolism

Associated products

  • Alternative Versions

    • Anti-Caspase-9 antibody [E23] - BSA and Azide free (ab219590)
    • PE Anti-Caspase-9 antibody [E23] (ab237431)
  • Assay kits

    • Caspase 9 Assay Kit (Colorimetric) (ab65608)
    • Caspase 9 (active) FITC Staining Kit (ab65615)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • HeLa whole cell lysate (ab150035)
    • HeLa whole cell lysate (ab29545)
    • Jurkat whole cell lysate (ab30128)
    • Jurkat whole cell lysate (ab7899)
  • Recombinant Protein

    • Recombinant human Caspase-9 protein (Tagged) (ab198061)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32539 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 (4)
1/1000 - 1/10000. Predicted molecular weight: 46 kDa.

We recommend overnight incubation at 4°C.

IHC-P
1/50 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Flow Cyt
1/100 - 1/250.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP
1/80 - 1/100.
ICC/IF
1/500.

For unpurified use at 1/50.

Notes
WB
1/1000 - 1/10000. Predicted molecular weight: 46 kDa.

We recommend overnight incubation at 4°C.

IHC-P
1/50 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Flow Cyt
1/100 - 1/250.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP
1/80 - 1/100.
ICC/IF
1/500.

For unpurified use at 1/50.

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.
  • Target information above from: UniProt accession P55211 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 842 Human
    • Omim: 602234 Human
    • SwissProt: P55211 Human
    • Unigene: 329502 Human
    • 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 [E23] (ab32539)
      Western blot - Anti-Caspase-9 antibody [E23] (ab32539)

      Lanes:
      Lane 1: Wild-type HAP1 whole cell lysate (40 µg)
      Lane 2: CASP9 knockout HAP1 whole cell lysate (40 µg)
      Lane 3: HeLa whole cell lysate (40 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab32539 observed at 45 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab32539 was shown to specifically recognize Caspase-9 in wild type HAP1 cells along with additional cross-reactive bands. No band was observed when Caspase-9 knockout samples were examined. Wild-type and Caspase-9 knockout samples were subjected to SDS-PAGE. Ab32539 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [E23] (ab32539)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [E23] (ab32539)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue labelling Caspase-9 with purified ab32539 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Immunocytochemistry/ Immunofluorescence - Anti-Caspase-9 antibody [E23] (ab32539)
      Immunocytochemistry/ Immunofluorescence - Anti-Caspase-9 antibody [E23] (ab32539)

      Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Caspase-9 with purified ab32539 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

      Control 1: primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

      Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

    • Flow Cytometry - Anti-Caspase-9 antibody [E23] (ab32539)
      Flow Cytometry - Anti-Caspase-9 antibody [E23] (ab32539)

      Flow Cytometry analysis of K562 cells labelling Caspase-9 with purified ab32539 at 1/250 (red). Cells were fixed with 100% methanol. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

    • Immunoprecipitation - Anti-Caspase-9 antibody [E23] (ab32539)
      Immunoprecipitation - Anti-Caspase-9 antibody [E23] (ab32539)

      ab32539 (purified) at 1/80 immunoprecipitating Caspase-9 in HeLa whole cell lysate.

      Lane 1 (input): HeLa whole cell lysate (10µg)

      Lane 2 (+): ab32539 + HeLa whole cell lysate.

      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32539 in HeLa whole cell lysate.

      For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Western blot - Anti-Caspase-9 antibody [E23] (ab32539)
      Western blot - Anti-Caspase-9 antibody [E23] (ab32539)
      All lanes : Anti-Caspase-9 antibody [E23] (ab32539) at 1/1000 dilution (purified)

      Lane 1 : HeLa whole cell lysate - treated with Camptothecin
      Lane 2 : HeLa whole cell lysate - untreated

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

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



      Blocking and dilution buffer: 5% NFDM/TBST.

    • Flow Cytometry - Anti-Caspase-9 antibody [E23] (ab32539)
      Flow Cytometry - Anti-Caspase-9 antibody [E23] (ab32539)

      Overlay histogram showing K562 cells stained with unpurified ab32539 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab32539, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was anti-rabbit DyLight® 488 (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in K562 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween used under the same conditions.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [E23] (ab32539)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [E23] (ab32539)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Caspase 9 with unpurified ab32539 at a dilution of 1/50.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    • Western blot - Anti-Caspase-9 antibody [E23] (ab32539)
      Western blot - Anti-Caspase-9 antibody [E23] (ab32539)
      All lanes : Anti-Caspase-9 antibody [E23] (ab32539) at 1/20000 dilution (unpurified)

      Lane 1 : Jurkat cell lysate - untreated
      Lane 2 : Jurkat cell lysate - treated with Camptothecin

      Predicted band size: 46 kDa
      Observed band size: 48 kDa why is the actual band size different from the predicted?
      Additional bands at: 28 kDa. We are unsure as to the identity of these extra bands.

    • Anti-Caspase-9 antibody [E23] (ab32539)
      Anti-Caspase-9 antibody [E23] (ab32539)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (143)

    Publishing research using ab32539? Please let us know so that we can cite the reference in this datasheet.

    ab32539 has been referenced in 143 publications.

    • Jin P  et al. Salidroside inhibits apoptosis and autophagy of cardiomyocyte by regulation of circular RNA hsa_circ_0000064 in cardiac ischemia-reperfusion injury. Gene 767:145075 (2021). PubMed: 32858179
    • Sun X  et al. Effect of Loureirin B on Crohn's disease rat model induced by TNBS via IL-6/STAT3/NF-?B signaling pathway. Chin Med 15:2 (2020). PubMed: 31911815
    • Duan C  et al. Drp1 regulates mitochondrial dysfunction and dysregulated metabolism in ischemic injury via Clec16a-, BAX-, and GSH- pathways. Cell Death Dis 11:251 (2020). PubMed: 32312970
    • Xu J  et al. SPAG5-AS1 inhibited autophagy and aggravated apoptosis of podocytes via SPAG5/AKT/mTOR pathway. Cell Prolif 53:e12738 (2020). PubMed: 31957155
    • Li H  et al. Long noncoding RNA SNHG17 induced by YY1 facilitates the glioma progression through targeting miR-506-3p/CTNNB1 axis to activate Wnt/ß-catenin signaling pathway. Cancer Cell Int 20:29 (2020). PubMed: 32009853
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-5 of 5 Abreviews

    Western blot abreview for Anti-Caspase-9 antibody [E23]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (U87 cells)
    Gel Running Conditions
    Non-reduced Non-Denaturing (Native) (4-20%)
    Loading amount
    13 µg
    Treatment
    bevasizumab 0, 1 and 2mg/ml for 24hrs, 48hrs
    Specification
    U87 cells
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 11 2020

    Western blot abreview for Anti-Caspase-9 antibody [E23]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    25 µg
    Gel Running Conditions
    Reduced Denaturing
    Sample
    Mouse Tissue lysate - whole (liver)
    Specification
    liver
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 22 2014

    Western blot abreview for Anti-Caspase 9 antibody [E23]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Fadu)
    Loading amount
    30 µg
    Specification
    Fadu
    Treatment
    RT8gray for 48hrs+Metformin400mM
    Gel Running Conditions
    Reduced Denaturing
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Read More

    Abcam user community

    Verified customer

    Submitted Apr 24 2013

    Immunohistochemistry (Frozen sections) abreview for Anti-Caspase 9 antibody [E23]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Rat Tissue sections (brain)
    Specification
    brain
    Fixative
    Formaldehyde
    Permeabilization
    No
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1.5% · Temperature: 22°C
    Read More

    Dr. Nitsan Kozlovsky

    Verified customer

    Submitted Aug 04 2011

    Western blot abreview for Anti-Caspase 9 antibody [E23]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (Brain lysate)
    Loading amount
    50 µg
    Specification
    Brain lysate
    Gel Running Conditions
    Reduced Denaturing
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Aug 02 2010

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