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.

    Some customers have used this antibody successfully in IHC-P however our latest tests were unsuccessful and therefore we can no longer guarantee this application. We would recommend ab32351 and ab184787 as an alternative product for this application.

  • Tested applications
    Suitable for: ICC/IF, IHC-Fr, WB, Flow Cyt, ICC, 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
    • 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.
  • General notes

      

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-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 - 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

  • 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.

  • 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 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 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:
  • Zhao W  et al. Long noncoding RNA NSCLCAT1 increases non-small cell lung cancer cell invasion and migration through the Hippo signaling pathway by interacting with CDH1. FASEB J 33:1151-1166 (2019). Read more (PubMed: 30148675) »
  • Payne SL  et al. Initial cell maturity changes following transplantation in a hyaluronan-based hydrogel and impacts therapeutic success in the stroke-injured rodent brain. Biomaterials 192:309-322 (2019). Read more (PubMed: 30468998) »
See all 424 Publications for this product

Customer reviews and Q&As

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1-10 of 44 Abreviews

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

Mr. Ehsan Pashay Ahi

Verified customer

Submitted Jul 28 2014

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Monkey Tissue sections (brain)
Antigen retrieval step
None
Permeabilization
Yes - triton 0.5%
Specification
brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5 · Temperature: 20°C
Fixative
Formaldehyde

Mrs. Francoise Geffroy

Verified customer

Submitted Mar 07 2019

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Mice Hepatocytes)
Gel Running Conditions
Reduced Denaturing (4-15% SDS-PAGE)
Loading amount
25 µg
Specification
Mice Hepatocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Dr. Armen Petrosyan

Verified customer

Submitted Feb 12 2019

Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Rat hepatocytes)
Permeabilization
Yes - 0.2% Triton X-100 in PBS
Specification
Rat hepatocytes
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
Fixative
Formaldehyde

Dr. Armen Petrosyan

Verified customer

Submitted Jun 29 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Liver)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 100X Citrate Buffer pH 6.0 (ab94674)
Permeabilization
Yes - 0.05% tween 20
Specification
Liver
Blocking step
Sea Block as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 22°C
Fixative
10%NBF

Mr. David Ivancic

Verified customer

Submitted Jun 08 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (RPMI - myeloma cell line)
Gel Running Conditions
Reduced Denaturing (8% SDS-PAGE)
Loading amount
25 µg
Specification
RPMI - myeloma cell line
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Dr. Armen Petrosyan

Verified customer

Submitted Jun 04 2018

Application
Western blot
Sample
Chicken Tissue lysate - whole (Chick embryo brainstem)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris gel (NuPAGE))
Loading amount
25 µg
Specification
Chick embryo brainstem
Blocking step
Odyssey Blocking Buffer (LI-COR) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Forrest Weghorst

Verified customer

Submitted Nov 24 2017

Application
Western blot
Sample
Dog Cell lysate - whole cell (Madin Darby Canine Kidney Cells)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Loading amount
20 µg
Treatment
Cisplatin 10 ´M 48 Hours
Specification
Madin Darby Canine Kidney Cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jul 25 2017

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

Mrs. 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

Abcam user community

Verified customer

Submitted Oct 09 2015

1-10 of 44 Abreviews

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