Validated using a knockout cell line
Recombinant
RabMAb

Anti-Caspase-3 antibody [EPR18297] (ab184787)

Overview

  • Product name
    Anti-Caspase-3 antibody [EPR18297]
    See all Caspase-3 primary antibodies
  • Description
    Rabbit monoclonal [EPR18297] to Caspase-3
  • Host species
    Rabbit
  • Specificity
    ab184787 recognizes pro-Caspase 3 and potentially cross reacts with active caspases after apoptosis has been induced in wildtype cells and not Caspase 3 knockout cells
  • Tested applications
    Suitable for: WB, IHC-P, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment within Human active + pro Caspase 3 aa 1-200. The exact sequence is proprietary.
    Database link: P42574

  • Positive control
    • WB: Jurkat whole cell lysate and Jurkat whole cell lysate treated with 1uM staurosporine for 4 hours. IHC-P: Human tonsil and cervical cancer tissues. IP: HeLa whole cell lysate treated with 1uM staurosporine 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 ab184787 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 1/2000. Detects a band of approximately 32, 17 kDa (predicted molecular weight: 32 kDa).

The 17 kDa band is the active form of the cleaved caspase 3 (subunit p17).
ab184787 recognizes pro-Caspase 3 and unable to detect the active caspases after induction in mouse and rat samples.

IHC-P 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
IP 1/80.

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.
  • 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
    • A830040C14Rik antibody
    • Apopain antibody
    • CASP-3 antibody
    • CASP3 antibody
    • CASP3_HUMAN antibody
    • Casp3a antibody
    • Caspase 3 antibody
    • Caspase 3, apoptosis-related cysteine peptidase antibody
    • Caspase 3, apoptosis-related cysteine protease antibody
    • Caspase 3, apoptosis-related cysteine protease a antibody
    • Caspase-3 subunit p12 antibody
    • CC3 antibody
    • CPP-32 antibody
    • CPP32 antibody
    • CPP32B antibody
    • Cysteine protease CPP32 antibody
    • EC 3.4.22.56 antibody
    • LICE antibody
    • mldy antibody
    • OTTHUMP00000165052 antibody
    • OTTHUMP00000165053 antibody
    • OTTHUMP00000165054 antibody
    • PARP cleavage protease antibody
    • Procaspase3 antibody
    • Protein Yama antibody
    • SCA 1 antibody
    • SCA-1 antibody
    • SREBP cleavage activity 1 antibody
    • Yama antibody
    see all

Images

  • Lane 1: Wild-type HAP1 cell lysate + Staurosporine (1μM for 4h)
    Lane 2: Wild-type HAP1 cell lysate
    Lane 3: Caspase-3 knockout HAP1 cell lysate + Staurosporine (1μM for 4h)
    Lane 4: Caspase-3 knockout HAP1 cell lysate
    Lanes 1 - 4: Merged signal (red and green). Green - ab184787 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab184787 was shown to recognise pro Caspase 3 when Caspase 3 knockout samples were used, along with additional cross-reactive bands. Wild-type and Caspase 3 knockout samples (± Staurosporine treatment) were subjected to SDS-PAGE. ab184787 and ab8245 (loading control to GAPDH) were diluted to 1/2000 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 hour at room temperature before imaging.

  • All lanes : Anti-Caspase-3 antibody [EPR18297] (ab184787) at 0.7 µg/ml

    Lane 1 : Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) treated with 1µM Staurosporine for 4 hours whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 32 kDa
    Observed band size: 32 kDa



    Blocking and diluting buffer: 5% NFDM/TBST.

    ab184787 recognizes pro-Caspase 3 and unable to detect the active caspases after induction in mouse and rat samples.

  • All lanes : Anti-Caspase-3 antibody [EPR18297] (ab184787) at 1/2000 dilution

    Lane 1 : Untreated Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates
    Lane 2 : Jurkat whole cell lysates treated with 1uM staurosporine for 4 hours

    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: 32 kDa
    Observed band size: 17,32 kDa (why is the actual band size different from the predicted?)


    Exposure time: 1 minute


    Blocking/Dilution buffer: 5% NFDM/TBST.

    Specificity: interacts with full length pro-Caspase 3 and the p17 subunit.

    The Caspase-3 precursor is first cleaved between D175 and S176 to produce the p11 subunit and p20 fragment. Subsequently, the p20 fragment is cleaved between D28 and S29 to generate the p17 subunit (Proc. Natl. Acad. Sci. USA. 93, 7464-7469 - PMID:8755496).

  • All lanes : Anti-Caspase-3 antibody [EPR18297] (ab184787) at 1/2000 dilution

    Lane 1 : Mouse brain tissue lysate
    Lane 2 : Mouse spleen tissue lysate
    Lane 3 : Rat spleen tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 32 kDa
    Observed band size: 32 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab184787 observed at 32 kDa. Red - loading control, ab18058, observed at 130 kDa.

     

    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab184787 and ab18058 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

  • Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling active and pro Caspase 3 with ab184787 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus and cytoplasm staining on lymphocytes of tonsil 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.

  • Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labeling active and pro Caspase 3 with ab184787 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus and cytoplasm staining on tumor cells of Human cervix cancer 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.

  • active and pro Caspase 3 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated with 1uM staurosporine for 4 hours with ab184787 at 1/80 dilution. Western blot was performed from the immunoprecipitate using ab184787 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used as secondary antibody at 1/1500 dilution.

    Lane 1: HeLa whole cell lysate treated with 1uM staurosporine for 4 hours 10 µg (Input).

    Lane 2: ab184787 IP in HeLa whole cell lysate treated with 1uM staurosporine for 4 hours.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab184787 in HeLa whole cell lysate treated with 1uM staurosporine for 4 hours.
    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 3 minutes.

References

This product has been referenced in:
  • Ankay Yilbas A  et al. Procaine and saline have similar effects on articular cartilage and synovium in rat knee. BMC Anesthesiol 18:51 (2018). IHC-P ; Rat . Read more (PubMed: 29743011) »
  • Zou Y  et al. LncRNA OIP5-AS1 regulates radioresistance by targeting DYRK1A through miR-369-3p in colorectal cancer cells. Eur J Cell Biol 97:369-378 (2018). WB ; Human . Read more (PubMed: 29773344) »

See all 2 Publications for this product

Customer reviews and Q&As

Application
Western blot
Sample
Human Cell lysate - whole cell (Glioma)
Gel Running Conditions
Reduced Denaturing (12.5% gel)
Loading amount
25 µg
Specification
Glioma
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted May 23 2016

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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