Overview

  • Product name
  • Description
    Rabbit polyclonal to Caspase-3
  • Host species
    Rabbit
  • Specificity
    ab13847 recognizes a cleaved form of Caspase 3 (~17 kDa) after apoptosis has been induced in wildtype cells and not Caspase 3 knockout cells.
  • Tested applications
    Suitable for: ICC/IF, IHC-P, IHC-Fr, WB, Flow Cyt, ICC, IHC (PFA fixed), IHC-FoFr, IHC - Wholemountmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Pig, Xenopus laevis, Drosophila melanogaster, Indian muntjac, Zebrafish, Rhesus monkey, Common marmoset, Schmidtea mediterranea, Salvelinus alpinus
    Predicted to work with: Dog, Chinese hamster
  • Immunogen

    Synthetic peptide corresponding to Human Caspase-3 aa 150-250 (internal sequence) conjugated to keyhole limpet haemocyanin.
    Database link: P42574
    (Peptide available as ab13848)

  • Positive control
    • IHC-P: Mouse embryo and liver tissue; Juvenile marmoset testis tissue. WB: HAP1 cell lysate; human caspase-3 recombinant protein. Flow Cyt: Jurkat cells. IHC-Fr: Tumour tissue; Rat brain tissue with a kainite lesion. ICC/IF: HeLa cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab13847 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
ICC/IF Use a concentration of 5 µg/ml.
IHC-P 1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-Fr Use at an assay dependent concentration.
WB 1/500. Detects a band of approximately 17, 34 kDa (predicted molecular weight: 17, 34 kDa).
Flow Cyt 1/500.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

ICC Use at an assay dependent concentration.
IHC (PFA fixed) 1/300.
IHC-FoFr 1/300.
IHC - Wholemount 1/500.

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

  • WT and cKO embryos cultured from 8.5–9.5 dpc in the presence (+VEGF) or absence (−VEGF) of VEGF.

    Embryos and yolk sacs were dissected and fixed in 4% paraformaldehyde (PFA)/PBS overnight at 4°C. The following day they were washed in PBS, dehydrated in an ascending methanol sequence, xylene treated, embedded in paraffin and sectioned at 7.5 µm.

    For immunofluorescence (IF) slides were dewaxed in xylenes, rehydrated with EtOH, and subjected to antigen retrieval in Tris buffer pH 10.0 for 10 min. The slides were then washed in PBT and incubated in blocking buffer (0.5% milk powder, 99.5% PBT) for 2 hrs at room temperature and then with primary antibody (caspase-3, 1/500 dilution; CDH1, 1/500 dilution) in blocking buffer at 4°C overnight in a humid chamber. Slides were then washed three times with PBT, incubated for 1 hr with secondary antibody in blocking buffer at room temperature. Nuclei were countered stained with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes, 1∶10,000) for 3 min and then coverslipped with Prolong Gold Antifade Reagent (Invitrogen). Sections were imaged on a Nikon Eclipse TE2000-S inverted microscope with Retiga EXi Fast camera with NIS Elements imaging software.

    Immunofluorescence against cleaved Caspase-3 (CASP3, green) and CDH1 (red) of sectioned yolk sacs revealed that typical CDH1 expression found in WT (U) and WT cultured with VEGF (V), was downregulated in cultured cKO embryos but more normal visceral endoderm expression restored when cKO embryos were cultured with VEGF (X).

  • Confocal immunofluorescence microscopy of caspase 3 (red fluorescence) using an antibody detecting pro-caspase 3 recombinant protein (ab13847) with DAPI-counterstained nuclei (blue fluorescence) in liver sections of control or ACF adult rats.

    Liver sections were incubated overnight with primary antibodies. After incubation with primary antibodies, the tissue sections were washed with PBS and then incubated with red fluorescent Alexa Fluor 594 donkey anti-rabbit antibody. Finally, the tissue sections were washed in PBS, mounted on vectashield (Vector Laboratories) and imaged on a confocal laser scanning microscope, LSM510, equipped with an argon laser (458/488/514 nm), a green helium/neon laser (543 nm), and a red helium/neon laser (633 nm; Carl Zeiss, Göttingen, Germany). Single optical slice images were taken using x10 or x20 Plan-Neofluar air interface or x40 Plan-Neofluar oil interface objective lens.

    Caspase 3 immunoreactivity was confined primarily to the well defined subcellular organelle-like structures in hepatic cells of control rats (A, B). In contrast in ACF rats, caspase 3 immunofluorescence was transferred to the perinuclear area of cells or inside nuclei of hepatic cells indicating an activation of pro-apoptotic factor caspase 3 (C,D).

    Bars = 20 μm.

  • Lane 1: Wild-type HAP1 cell lysate + Staurosporine (ab146588)  (1μM for 4h)
    Lane 2: Wild-type HAP1 cell lysate
    Lane 3: Caspase-3 knockout HAP1 cell lysate + Staurosporine (ab146588) (1μM for 4h)
    Lane 4: Caspase-3 knockout HAP1 cell lysate

    Lanes 1 - 4: Merged signal (red and green). Green - ab13847 observed at 17 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab13847 was shown to recognise 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. ab13847 and ab8245 (loading control to GAPDH) were diluted to 1/500 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.

  • ab13847 staining active caspase 3 in Human Jurkat cells by Flow Cytometry. Cells were prepared in a phosphate buffered solution containing 0.1% sodium azide with FBS fixed with paraformaldehyde and permeabilized with Triton X-100 and NP40. The sample was incubated with the primary antibody (1/100 in wash buffer) for 24 hours at 4°C. A FITC-conjugated Goat anti-rabbit Ig (1/100) was used as the secondary antibody.

    Gating Strategy: Isolate cell population from plot of SSC-A / FSA-A

  • IHC-P image of Caspase 3 staining with ab13847 on tissue sections from juvenile marmoset testis. The sections were subjected to heat-mediated antigen retrieval using Dako antigen retrieval solution. The sections were then blocked with 5% milk for 30 minutes at 25°C, before incubation with ab13847 (1/100 dilution) for 18 hours at 4°C. The secondary was an Alexa-Fluor 555 conjugated goat anti-rabbit polyclonal, used at a 1/500 dilution.

    See Abreview

  • 5 μm frozen sections of tumor tissue were fixed with 100% ice cold methanol for 10 minutes, then blocked in 5% normal goat serum in PBS (pH 7.4) for 1h. Sections were incubated with ab13847 (1:500) at 4°C overnight and for 1h with secondary antibodies at room temperature.

  • All lanes : Anti-Caspase-3 antibody (ab13847) at 1 µg/ml

    Lane 1 : Human Caspase 3 (active) Recombinant Protein
    Lane 2 : Human Pro Caspase 3 (inactive) Recombinant Protein

    Lysates/proteins at 0.1 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 17, 34 kDa
    Observed band size: 17,32 kDa (why is the actual band size different from the predicted?)


    Exposure time: 8 minutes


    Caspase 3 exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce large (17kDa) and small (12kDa) subunits. These subunits dimerize to form the active enzyme. ab13847 specifically detects the large active subunit (17kDa) and the  inactive pro Caspase 3 (32 kDa).

    This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab13847 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

     

    Secondary antibody - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody

  • ab13847 was used in IHC of frozen sections from a rat brain with a kainite lesion. The non lesionned contralateral site serves as a negative control. The sections were fixed with paraformaldehyde. The tissue was perfused with 4% PFA and embedded in OCT compound and cut on the cryostat. The primary antibody was incubated for 12 hours at a dilution of 1/300. A biotin labelled secondary antibody was used at a dilution of 1/300.
  • HeLa cells were fixed for 10 minutes at room temperature in 3.7% PFA and permeabilised in 0.1% Triton X-100/PBS then incubated with ab13847 (5µg/ml) for 1 hour at room temperature. The top panel shows control cells treated with DMSO. The bottom panel shows HeLa cells treated with 1 mM staurosporine (ab146588) for 4 hours to induce caspase-3 activation. ab13847 staining is shown in green and counterstaining with DAPI is shown in blue. 100x magnification.

    The image shows the staining with ab13847 is very faint in the untreated control cultures,  but  very  bright  after  activation  of capsase-3 by treatment with  the staurosporine . (N.B. in these cultures the nuclei are apoptotic).

     
     

References

This product has been referenced in:
  • Gao C  et al. Neuroprotective effects of hydrogen sulfide on sodium azide-induced oxidative stress in PC12 cells. Int J Mol Med 41:242-250 (2018). Read more (PubMed: 29115393) »
  • Yu X  et al. Baicalein induces cervical cancer apoptosis through the NF-?B signaling pathway. Mol Med Rep 17:5088-5094 (2018). Read more (PubMed: 29393414) »

See all 236 Publications for this product

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