Anti-active + pro Caspase 3 antibody (ab13847)

Overview

  • Product nameAnti-active + pro Caspase 3 antibody
    See all active + pro Caspase 3 primary antibodies
  • Description
    Rabbit polyclonal to active + pro Caspase 3
  • SpecificityAb13847 has been batch tested in WB using recombinant pro-Caspase 3 and recombinant active Caspase 3 only. Not all batches may detect endogenous caspase 3 in WB. However, some customers have successfully used ab13847 on endogenous lysates.
  • 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, Fruit fly (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 active + pro 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.

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 1/1000 - 1/2000. Detects a band of approximately 17,32 kDa (predicted molecular weight: 17,32 kDa).Can be blocked with Human active Caspase-3 peptide (ab13848).

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).

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

Target

  • RelevanceCaspases are a family of cysteine proteases that are key mediators of programmed cell death or apoptosis. The precursor form of all caspases is composed of a prodomain, and large and small catalytic subunits. The active forms of caspases are generated by several stimuli including ligand receptor interactions, growth factor deprivation and inhibitors of cellular functions. All known caspases require cleavage adjacent to aspartates to liberate one large and one small subunit, which associate into a2b2 tetramer to form the active enzyme. Gene for Caspase 3 also known as Yama, CPP32, and apopain codes for a 32-kDa protein. Caspase 3 cleaves the death substrate poly(ADP ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis and is inhibitable by the CrmA protein. Other Caspase 3 substrates include DNA-PK, actin, GAS2, and procaspase 6, etc. Caspase 3 is activated by cleavage events at Asp-28/Ser-29 (between N-terminal pro domain) and Asp-175/Ser-176 (between large and small subunits) to generate a large subunit of 17 kDa and a small subunit of 12 kDa.
  • Cellular localizationCytoplasmic
  • Database links
  • Alternative names
    • APOPAIN antibody
    • Apopain precursor antibody
    • CASP3 antibody
    • Caspase 3 apoptosis related cysteine protease antibody
    • Caspase3 antibody
    • CPP 32 antibody
    • CPP32B antibody
    • Cysteine protease CPP32 antibody
    • Human cysteine protease CPP32 isoform alpha mRNA complete cds antibody
    • ICE3 antibody
    • LICE antibody
    • PARP cleavage protease antibody
    • SCA1 antibody
    • SREBP cleavage activity 1 antibody
    • Yama antibody
    • Yama protein antibody
    see all

Anti-active + pro Caspase 3 antibody images

  • 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 DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • ab13847 staining caspase 3 in SKNSH cells treated with Z-VDVAD-FMK (ab142035), by ICC/IF. Decrease in caspase 3 expression correlates with increased concentration of Z-VDVAD-FMK, as described in literature.
    The cells were incubated at 37°C for 1 hour in media containing different concentrations of ab142035 (Z-VDVAD-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 DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • ab13847 staining Drosophila melanogaster 3rd instar wing imaginal disc (red) by IHC-wholemount. The tissue was fixed with 4% formaldehyde in PBS at room temperature for 20 minutes. Sample was blocked in PBS+ Triton-X 100 0.3% + BSA 1% for 1h at room temperature, followed by staining with ab13847 at 1/500 (in PBS+ Triton-X 100 0.3% + BSA 1%) for 16h at 4°C. A Cy3 conjugated goat anti-rabbit polyclonal secondary antibody at 1/200 was incubated for 2h at room temperature. Nuclei were stained with DAPI.

    See Abreview

  • 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.
  • ab13847 at a dilution of 1/500 staining Asynchronous HeLa cells by Immunocytochemistry. The antibody was incubated with the cells for 30 minutes and then detected using a Cy3 conjugated Goat Anti-Rabbit IgG (H+L). This antibody gives a predominantly nuclear focal staining pattern in all interphase nuclei investigated.

    This image is courtesy of an Abreview by Kirk McManus submitted on 2 December 2005.

    See Abreview

  • 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).

  • 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

  • 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

  • All lanes : Anti-active + pro 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 : 17,32 kDa
    Observed band size : 17,32 kDa


    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.

  • ICC/IF image of ab13847 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab13847, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293 and MCF7 cells at 5µg/ml.
  • 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 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 DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • 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 DyLight 488 goat anti-rabbit polyclonal 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-active + pro Caspase 3 antibody (ab13847)

This product has been referenced in:
  • Roy MG  et al. Muc5b is required for airway defence. Nature 505:412-6 (2014). Mouse . Read more (PubMed: 24317696) »
  • Duan LJ  et al. Hypoxia inducible factor-2a regulates the development of retinal astrocytic network by maintaining adequate supply of astrocyte progenitors. PLoS One 9:e84736 (2014). ICC/IF ; Mouse . Read more (PubMed: 24475033) »

See all 60 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 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)
Blocking step Milk as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate buffer pH 6,0
Sample Fish Tissue sections (Ovary)
Specification Ovary
Permeabilization Yes - Triton
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

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

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