Overview

  • Product nameAnti-Caspase-3 antibody
    See all Caspase-3 primary antibodies
  • Description
    Rabbit polyclonal to Caspase-3
  • Specificityab13847 recognizes a cleaved form of Caspase 3 (~17 kDa) after apoptosis has been induced in wildtype cells and not Caspase 3 knockout cells
  • Tested applicationsICC/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, Marmoset (common), Schmidtea mediterranea, Salvelinus alpinus
    Predicted to work with: Dog, Chinese Hamster
  • Immunogen

    Synthetic peptide corresponding to Human Caspase-3 aa 150-250 conjugated to Keyhole Limpet Haemocyanin (KLH).
    (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

  • FunctionInvolved 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 specificityHighly 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 similaritiesBelongs 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 localizationCytoplasm.
  • 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

Anti-Caspase-3 antibody images



  • 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 (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 - 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 (± Stauroprine 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) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • ab13847 at 1/1000 staining Xenopus laevis wholemount and sectioned tails by IHC-P. The tissue was formaldehyde fixed and blocked prior to incubation with the antibody for 12 hours. An alkaline phosphatase conjugated goat anti-rabbit antibody was used as the secondary.

    See Abreview

  • 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.

  • 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

  • All lanes : Anti-Caspase-3 antibody (ab13847) at 1/500 dilution

    Lane 1 : HeLa Whole Cell Lysate
    Lane 2 : HeLa Staurosporine Treated (2 uM/4hr) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 17 kDa
    Observed band size : 16 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 100 kDa,25 kDa (possible cleavage fragment),27 kDa (possible cleavage fragment),40 kDa. We are unsure as to the identity of these extra bands.

    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 2% 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 ab133406

  • 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.

  • ab13847 staining Salvelinus alpinus (a member of Salmonidae) by whole-mount assay in a pre-hatching embryonic stage. The embryos fixed in 4% paraformaldehyde were kept in 100% methanol at -20C and then rehydrated by increasing dilution series of 1 x PBST (1 x PBS with 0.1% Tween). The embryos were dechorinated and treated with 20 to 40 ug/ml proteinase K ( for 22 to 60 min, depending on the relative-ages. Prior to the blocking step embryos were washed with PDT buffer (1 x PBST, 0.3% Triton X, 1% DMSO) for 1 hour at room temperature and then were transferred to blocking solution (10% heat-inactivated FBS, 8% BSA in PBST) for another hour at room temperature. A 1:500 dilution of anti-activated + pro Caspase-3 antibody (abcam, ab13847) was added and embryos were incubated at 4C overnight. A goat polyclonal secondary antibody to rabbit IgG - H&L (Alexa Fluor­ 488) (abcam, ab150077) was used for detection at 1:400 dilution and 4C overnight incubation. (A) Negative control with only the secondary antibody and (B) ab13847 stained embryo (Mo: mouth opening L: lower jaw).

    See Abreview

  • 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 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 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 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).

  • 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 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 for Anti-Caspase-3 antibody (ab13847)

This product has been referenced in:
  • Yu-Ching Lin Z  et al. Gene expression ontogeny of spermatogenesis in the marmoset uncovers primate characteristics during testicular development. Dev Biol N/A:N/A (2015). IHC-P ; Marmoset (common) . Read more (PubMed: 25624265) »
  • Hamilton A  et al. Loss of Serglycin Promotes Primary Tumor Growth and Vessel Functionality in the RIP1-Tag2 Mouse Model for Spontaneous Insulinoma Formation. PLoS One 10:e0126688 (2015). Read more (PubMed: 25978773) »

See all 77 Publications for this product

Product Wall

Application IHC - Wholemount
Sample Salvelinus alpinus Embryo (Developing head at a pre-hatching embryonic stage)
Specification Developing head at a pre-hatching embryonic stage
Username

Mr. Ehsan Pashay Ahi

Verified customer

Submitted Jul 28 2014

Application Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample Mouse Tissue sections (brain bearing human tumor)
Antigen retrieval step None
Permeabilization Yes - triton 0.2%
Specification brain bearing human tumor
Blocking step BSA 1% + Donkey serum 5% as blocking agent for 30 minute(s) · Temperature: 22°C
Fixative Paraformaldehyde
Username

Ms. Francoise Geffroy

Verified customer

Submitted Jul 28 2016

Application Western blot
Sample Mouse Tissue lysate - whole (Kidney)
Gel Running Conditions Reduced Denaturing (4-12%)
Loading amount 20 µg
Specification Kidney
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 Oct 09 2015

Application Western blot
Sample Rat Tissue lysate - whole (Heart)
Gel Running Conditions Reduced Denaturing (10%)
Loading amount 65 µg
Specification Heart
Blocking step BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Oct 06 2015

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 35°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate buffer
Sample Rat Tissue sections (Bone - Femur)
Specification Bone - Femur
Permeabilization No
Fixative Paraformaldehyde
Username

Mr. Helder Fonseca

Verified customer

Submitted Oct 06 2014

Application Western blot
Loading amount 1000 cells
Gel Running Conditions Non-reduced Non-Denaturing (Native)
Sample Mouse Cell lysate - whole cell (Brain)
Specification Brain
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Username

Abcam user community

Verified customer

Submitted Sep 23 2014

Application Immunohistochemistry (Frozen sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Sample Mouse Tissue sections (Brain)
Specification Brain
Permeabilization No
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 23 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Fish Tissue sections (Ovary)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate buffer pH 6,0
Permeabilization Yes - Triton
Specification Ovary
Blocking step Milk as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative Paraformaldehyde
Username

Dr. Monica Cassel

Verified customer

Submitted Jul 29 2014

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

Zachary Yu-Ching Lin

Verified customer

Submitted Aug 01 2013

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