Validated using a knockout cell line
Recombinant
RabMAb

Anti-active Caspase-3 antibody [E83-77] (ab32042)

Overview

  • Product name
    Anti-active Caspase-3 antibody [E83-77]
    See all active Caspase-3 primary antibodies
  • Description
    Rabbit monoclonal [E83-77] to active Caspase-3
  • Specificity
    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 antibody only detects the active (cleaved) form of Caspase-3 and does not recognize the pro form of Caspase-3. PubMedID 19789217 describes the detection of human cells injected into mice.
  • Tested applications
    Suitable for: IHC-Fr, WB, ICC/IFmore details
    Unsuitable for: Flow Cyt or IP
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human active Caspase-3 aa 1-100 (N terminal). A synthetic peptide corresponding to residues following Ser29 of human Caspase 3 (N terminus of p17 subunit).
    Database link: P42574

  • Positive control
    • WB: Jurkat cell lysate (camptothecin treated); HeLa cell lysate (staurosporine treated). IF: Hela cells (staurosporine treated).
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

     

    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 ab32042 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-Fr Use at an assay dependent concentration.
WB 1/500. Detects a band of approximately 17 kDa (predicted molecular weight: 32 kDa).
ICC/IF 1/100 - 1/250.
  • Application notes
    Is unsuitable for Flow Cyt or IP.
  • Target

    • Function
      Involved in the activation cascade of caspases responsible for apoptosis execution. At the onset of apoptosis it proteolytically cleaves poly(ADP-ribose) polymerase (PARP) at a '216-Asp-
      -Gly-217' bond. Cleaves and activates sterol regulatory element binding proteins (SREBPs) between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. Cleaves and activates caspase-6, -7 and -9. Involved in the cleavage of huntingtin. Triggers cell adhesion in sympathetic neurons through RET cleavage.
    • Tissue specificity
      Highly expressed in lung, spleen, heart, liver and kidney. Moderate levels in brain and skeletal muscle, and low in testis. Also found in many cell lines, highest expression in cells of the immune system.
    • Sequence similarities
      Belongs to the peptidase C14A family.
    • Post-translational
      modifications
      Cleavage by granzyme B, caspase-6, caspase-8 and caspase-10 generates the two active subunits. Additional processing of the propeptides is likely due to the autocatalytic activity of the activated protease. Active heterodimers between the small subunit of caspase-7 protease and the large subunit of caspase-3 also occur and vice versa.
      S-nitrosylated on its catalytic site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway, associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active site thiol.
    • Cellular localization
      Cytoplasm.
    • Information by UniProt
    • Database links
    • Alternative names
      • Apopain antibody
      • CASP 3 antibody
      • CASP-3 antibody
      • CASP3 antibody
      • CASP3_HUMAN antibody
      • Caspase 3 antibody
      • Caspase-3 subunit p12 antibody
      • cleaved caspase 3 antibody
      • CPP 32 antibody
      • CPP-32 antibody
      • CPP32B antibody
      • Cysteine protease CPP32 antibody
      • PARP cleavage protease antibody
      • Protein Yama antibody
      • SCA-1 antibody
      • SCA1 antibody
      • SREBP cleavage activity 1 antibody
      • Yama antibody
      see all

    Images



    • Predicted band size : 32 kDa

      Lane 1: Wild type HAP1 + DMSO for 24 hours, whole cell lysate (20 µg)
      Lane 2: Wild type HAP1 + 2uM Staurosporine (ab146588) for 24 hours, whole cell lysate (20 µg)
      Lane 3: HAP1 CASP3 KO + DMSO for 24 hours, whole cell lysate (20 µg)
      Lane 4: HAP1 CASP3 KO + 2uM Staurosporine (ab146588) for 24 hours, whole cell lysate (20 µg)
      Lane 5: HeLa + DMSO for 24 hours, whole cell lysate (20 µg)
      Lane 6: HeLa + 2uM Staurosporine (ab146588) for 24 hours, whole cell lysate (20 µg)
      Lanes 1 - 6: Merged signal (red and green). Green - ab32042 observed at 17 kDa. Red - loading control, ab8245, observed at 130 kDa.

      ab32042 was shown to specifically react with CASP3 (Caspase-3) when CASP3 (Caspase-3) knockout samples were used. HAP1 wild-type and CASP3 (Caspase-3) knockout samples were subjected to SDS-PAGE. Ab32042 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 500 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

      Cells were grown to confluency prior to treatment.

    • Ab32042, at dilution of 1/100, staining HeLa cells by Immunofluorescence.
      Left image: control.
      Right image: staurosporine treated.
    • All lanes : Anti-active Caspase-3 antibody [E83-77] (ab32042) at 1/500 dilution

      Lane 1 : HeLa Whole Cell Lysate (2 uM Staurosporine, 4Hr) at 20 µg
      Lane 2 : HeLa Whole Cell Lysate (untreated) at 20 µg
      Lane 3 : Active Caspase 3 (recombinant protein) at 0.1 µg

      Secondary
      800CW Goat Anti-Rabbit IgG at 1/10000 dilution

      Performed under reducing conditions.

      Predicted band size : 32 kDa
      Additional bands at : 17 kDa (possible mature (processed) protein).
    • Lanes 1 - 2 : anti Pro Caspase 3 at 1/10000 dilution
      Lanes 3 - 4 : Anti-active Caspase-3 antibody [E83-77] (ab32042) at 1/500 dilution

      Lane 1 : Jurkat cell lysate
      Lane 2 : Jurkat cell lysate + Camptothecin
      Lane 3 : Jurkat cell lysate
      Lane 4 : Jurkat cell lysate + Camptothecin


      Predicted band size : 32 kDa
      Observed band size : 17 kDa (why is the actual band size different from the predicted?)
      Additional bands at : 30 kDa. We are unsure as to the identity of these extra bands.

    Protocols

    References

    This product has been referenced in:
    • Deng YP  et al. Human bocavirus induces apoptosis and autophagy in human bronchial epithelial cells. Exp Ther Med 14:753-758 (2017). Read more (PubMed: 28672995) »
    • Wei M  et al. Knockdown of RNF2 induces cell cycle arrest and apoptosis in prostate cancer cells through the upregulation of TXNIP. Oncotarget 8:5323-5338 (2017). IHC . Read more (PubMed: 28029659) »

    See all 47 Publications for this product

    Customer reviews and Q&As

    Filter by Application

    Filter by Species

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    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (BT-474 (Breast Cancer Cell Line))
    Permeabilization
    Yes - 0.1% Triton in PBS
    Specification
    BT-474 (Breast Cancer Cell Line)
    Blocking step
    3% BSA + 5% Goat Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 26°C
    Fixative
    Paraformaldehyde
    Username

    Abcam user community

    Verified customer

    Submitted Oct 18 2017

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Monkey Cell (Kidney)
    Permeabilization
    Yes - 0.1% triton x-100
    Specification
    Kidney
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
    Fixative
    Paraformaldehyde
    Username

    Abcam user community

    Verified customer

    Submitted Jul 29 2016

    Abcam has not validated the combination of species/application used in this Abreview.
    Application
    Western blot
    Sample
    Monkey Cell lysate - whole cell (Kidney)
    Gel Running Conditions
    Non-reduced Non-Denaturing (Native)
    Loading amount
    30 µg
    Specification
    Kidney
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
    Username

    Abcam user community

    Verified customer

    Submitted May 06 2016

    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Brain)
    Permeabilization
    Yes - 0.3% Triton x 100: 30 min
    Specification
    Brain
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
    Fixative
    Paraformaldehyde
    Username

    Abcam user community

    Verified customer

    Submitted May 06 2016

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (prostate cancer)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: 0.05% trysin
    Permeabilization
    Yes - 0.1% Triton X-100
    Specification
    prostate cancer
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 23°C
    Fixative
    Formaldehyde
    Username

    Abcam user community

    Verified customer

    Submitted Dec 24 2015

    Abcam has not validated the combination of species/application used in this Abreview.
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Rat Tissue sections (Thymus)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citric acid
    Permeabilization
    No
    Specification
    Thymus
    Blocking step
    BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
    Fixative
    Formaldehyde
    Username

    Mr. Carl Hobbs

    Verified customer

    Submitted Dec 08 2015

    Abcam has not validated the combination of species/application used in this Abreview.
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Human melanoma xenograft)
    Antigen retrieval step
    Heat mediated
    Permeabilization
    No
    Specification
    Human melanoma xenograft
    Blocking step
    BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
    Fixative
    Formaldehyde
    Username

    Mr. Carl Hobbs

    Verified customer

    Submitted Dec 08 2015

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Liver)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: EDTA pH9
    Permeabilization
    No
    Specification
    Liver
    Blocking step
    BSA as blocking agent for 20 minute(s) · Concentration: 1% · Temperature: 25°C
    Fixative
    Formaldehyde
    Username

    Abcam user community

    Verified customer

    Submitted Aug 13 2015

    Application
    Western blot
    Loading amount
    50 µg
    Gel Running Conditions
    Reduced Denaturing
    Sample
    Human Cell lysate - whole cell (HT29)
    Specification
    HT29
    Treatment
    IGF-1R inhibitor
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

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    Verified customer

    Submitted Mar 21 2014

    Application
    Western blot
    Loading amount
    20 µg
    Gel Running Conditions
    Reduced Denaturing (12%)
    Sample
    Rabbit Cell lysate - whole cell (lung)
    Specification
    lung
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

    Abcam user community

    Verified customer

    Submitted Sep 24 2013

    1-10 of 18 Abreviews

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