Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Caspase-3 antibody [EPR18297] - BSA and Azide free (ab224271)

Overview

  • Product name

    Anti-Caspase-3 antibody [EPR18297] - BSA and Azide free
    See all Caspase-3 primary antibodies
  • Description

    Rabbit monoclonal [EPR18297] to Caspase-3 - BSA and Azide free
  • Host species

    Rabbit
  • Specificity

    ab224271 recognizes pro-Caspase 3 and cross reacts with active caspases after apoptosis has been induced in wildtype cells and not Caspase 3 knockout cells
  • Tested applications

    Suitable for: IP, WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment 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

    Ab224271 is the carrier-free version of ab184787. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    ab224271 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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 ab224271 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
IP Use at an assay dependent concentration.
WB Use at an assay dependent concentration. 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).

IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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
    • 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
    • Caspase3 antibody
    • CC3 antibody
    • CPP 32 antibody
    • CPP-32 antibody
    • CPP32 antibody
    • CPP32B antibody
    • Cysteine protease CPP32 antibody
    • EC 3.4.22.56 antibody
    • ICE3 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
    • SCA1 antibody
    • SREBP cleavage activity 1 antibody
    • Yama antibody
    • Yama protein antibody
    see all

Images

  • This WB data was generated using the same anti-Caspase-3 antibody clone [EPR18297] in a different buffer formulation (cat# ab184787).

    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.

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184787).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184787).

  • 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 Detection Reagent (HRP) (ab131366) was used for detection 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184787).

References

ab224271 has not yet been referenced specifically in any publications.

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