Overview

  • Product name
  • Description
    Rabbit polyclonal to Caspase-3
  • 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.
    (Peptide available as ab13848)

  • Positive control
    • This antibody gave a positive signal in WB with active Caspase 3 recombinant protein and pro-Caspase 3 recombinant protein. This antibody also gave a signal with HeLa staurosporine treated (2uM/4 hr) whole cell lysate.

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 kDa (predicted molecular weight: 17 kDa).Can be blocked with Human Caspase-3 peptide (ab13848).
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



  • Performed under reducing conditions.

    Predicted band size : 17 kDa
    Observed band size : 26,30 kDa (why is the actual band size different from the predicted?)

    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.

  • 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

  • ab13847 staining caspase 3 in HeLa cells treated with RTIL-13™ (ab120465), by ICC/IF. Increase in caspase 3 expression correlates with increased concentration of RTIL-13™, as described in literature.
    The cells were incubated at 37°C for 24h in media containing different concentrations of ab120465 (RTIL-13™) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab13847 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A goat anti-rabbit DyLight 488 secondary antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • 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

  • ab13847 staining caspase 3 in SKNSH cells treated with Z-IETD-FMK (ab141382), by ICC/IF. Decrease in caspase 3 expression correlates with increased concentration of Z-IETD-FMK, as described in literature.
    The cells were incubated at 37°C for 1 hour in media containing different concentrations of ab141382 (Z-IETD-FMK) in DMSO. After this incubation, 10 μM of camptothecin (ab120115) was added to all samples and the cells were incubated for further 24 hours. The samples were then fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab13847 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A goat anti-rabbit DyLight 488 (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • 5μm 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
    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 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 HRP (ab97051)

  • 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).

     
     
  • ab13847 staining caspase 3 in A549 cells treated with quercetin (ab120247), by ICC/IF. Increase in caspase 3 expression correlates with increased concentration of quercetin, as described in literature.
    The cells were incubated at 37°C for 6h in media containing different concentrations of ab120247 (quercetin) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab13847 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A goat anti-rabbit DyLight 488 polyclonal secondary antibody (ab968999) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

References

This product has been referenced in:
  • Wang J  et al. Menin mediates Tat-induced neuronal apoptosis in brain frontal cortex of SIV-infected macaques and in Tat-treated cells. Oncotarget 8:18082-18094 (2017). WB, Functional Studies ; Rhesus monkey . Read more (PubMed: 28178646) »
  • He X  et al. Atorvastatin protects against contrast-induced nephropathy via anti-apoptosis by the upregulation of Hsp27 in vivo and in vitro. Mol Med Rep 15:1963-1972 (2017). WB . Read more (PubMed: 28260077) »

See all 168 Publications for this product

Customer reviews and Q&As

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (10)
Sample
Schmidtea mediterranea Tissue lysate - whole (whole planarians)
Specification
whole planarians
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted Sep 23 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Dako antigen retrieval solution
Sample
Marmoset (common) Tissue sections (Juvenile testis)
Specification
Juvenile testis
Permeabilization
No
Fixative
Formaldehyde
Username

煜欽 林

Verified customer

Submitted Aug 01 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
IHC - Wholemount
Sample
Fruit fly (Drosophila melanogaster) Tissue (wing disc)
Specification
wing disc
Username

Abcam user community

Verified customer

Submitted Jul 26 2013

Application
IHC - Wholemount
Sample
Zebrafish Embryo (Wholemount 3-5 days post fertilisation)
Specification
Wholemount 3-5 days post fertilisation
Username

Dr. Chrissy Hammond

Verified customer

Submitted Jul 23 2013

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (12.5% SDS-PAGE)
Sample
Human Cell lysate - whole cell (Colon cancer cell)
Specification
Colon cancer cell
Treatment
20uM WIN 55,212-2
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 16 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Sample
Mouse Cell (mouse Embryonic Stem Cell)
Specification
mouse Embryonic Stem Cell
Permeabilization
Yes - 0.3% PBST, 10 min
Fixative
Formaldehyde
Username

Feinstein Paul G

Verified customer

Submitted Jul 02 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 37°C
Sample
Human Cell (Caco-2)
Specification
Caco-2
Permeabilization
Yes - PBS 1X, Triton 1%
Fixative
Formaldehyde
Username

Dr. Pierre-Jean FERRON

Verified customer

Submitted Apr 23 2013

Thank you for contacting Abcam.

I managed to find antibodies against rat GFAP, FOX3, Iba1, and CD68 all raised in different hosts (chicken, rabbit, goat, and mouse, respectively). They each are highly rated by other researchers and have numero...

Read More
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
Sample
Fruit fly (Drosophila melanogaster) Cell (irradiated Drosophila larval eye imaginal disc)
Specification
irradiated Drosophila larval eye imaginal disc
Permeabilization
Yes - 0.3% Triton X 100
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 11 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric Buffer
Sample
Pig Tissue sections (Brain)
Specification
Brain
Permeabilization
No
Fixative
Paraformaldehyde
Username

Dr. Mariya Hristova

Verified customer

Submitted Mar 22 2012

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