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  1. Link

    pro-caspase-3-antibody-e83-103-ab32499.pdf

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Cell Biology Apoptosis Intracellular Caspases etc Caspases
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-pro Caspase-3 antibody [E83-103] (ab32499)

  • Datasheet
  • SDS
Submit a review Q&A (1)References (66)

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Western blot - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
  • Immunocytochemistry/ Immunofluorescence - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
  • Flow Cytometry (Intracellular) - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
  • Western blot - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
  • Anti-pro Caspase-3 antibody [E83-103] (ab32499)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E83-103] to pro Caspase-3
  • Suitable for: ICC/IF, WB, IHC-P, Flow Cyt (Intra)
  • Knockout validated
  • Reacts with: Mouse, Human

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Overview

  • Product name

    Anti-pro Caspase-3 antibody [E83-103]
    See all pro Caspase-3 primary antibodies
  • Description

    Rabbit monoclonal [E83-103] to pro Caspase-3
  • Host species

    Rabbit
  • Specificity

    This antibody only detects pro-form (35kD) of caspase-3, and does not recognize any cleaved caspases.

  • Tested applications

    Suitable for: ICC/IF, WB, IHC-P, Flow Cyt (Intra)more details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide corresponding to Human pro Caspase-3 (N terminal).

  • Positive control

    • Jurkat whole cell lysate (ab7899) and human colon adenocarcinoma.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    E83-103
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Caspases etc
    • Caspases

Associated products

  • Alternative Versions

    • Anti-pro Caspase-3 antibody [E83-103] - BSA and Azide free (ab238440)
  • Assay kits

    • Caspase-3 Assay Kit (Colorimetric) (ab39401)
    • Cleaved Caspase-3 Staining Kit (FITC) (ab65613)
  • Compatible Secondaries

    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • Jurkat whole cell lysate (ab30128)
    • Jurkat whole cell lysate (ab7899)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32499 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.
WB
1/10000. Detects a band of approximately 35 kDa (predicted molecular weight: 31 kDa).
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)
1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Notes
ICC/IF
Use a concentration of 5 µg/ml.
WB
1/10000. Detects a band of approximately 35 kDa (predicted molecular weight: 31 kDa).
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)
1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Relevance

    Caspases 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 localization

    Cytoplasmic
  • Database links

    • Entrez Gene: 836 Human
    • Entrez Gene: 12367 Mouse
    • Omim: 600636 Human
    • SwissProt: P42574 Human
    • SwissProt: P70677 Mouse
    • Alternative names

      • CASP3 antibody
      • Caspase 3 antibody
      • Caspase 3 apoptosis related cysteine peptidase antibody
      • CPP32 antibody
      • CPP32B antibody
      • Procaspase 3 antibody
      • SCA 1 antibody
      • SCA1 antibody
      • Yama antibody
      • Yama protein antibody
      see all

    Images

    • Western blot - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
      Western blot - Anti-pro Caspase-3 antibody [E83-103] (ab32499)

      Lane 1: Wild-type HAP1 cell lysate
      Lane 2: Wild-type HAP1 cell lysate + Staurosporine (1μM for 4h)
      Lane 3: Caspase-3 knockout HAP1 cell lysate
      Lane 4: Caspase-3 knockout HAP1 cell lysate + Staurosporine (1μM for 4h)
      Lanes 1 - 4: Merged signal (red and green). Green - ab32499 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab32499 was shown to specifically react with pro Caspase 3 when Caspase 3 knockout samples were used. Wild-type and Caspase 3 knockout samples (± Staurosporine treatment) were subjected to SDS-PAGE. ab32499 and ab8245 (loading control to GAPDH) were diluted to 1/1000 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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pro Caspase-3 antibody [E83-103] (ab32499)

      Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma ab32499 at 1/250 dilution.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Immunocytochemistry/ Immunofluorescence - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
      Immunocytochemistry/ Immunofluorescence - Anti-pro Caspase-3 antibody [E83-103] (ab32499)

      ICC/IF image of ab32499 stained HeLa cells. The cells were 4% formaldehyde 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 (ab32499, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, anti-rabbit DyLight® 488 used at a 1/250 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.

    • Flow Cytometry (Intracellular) - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
      Flow Cytometry (Intracellular) - Anti-pro Caspase-3 antibody [E83-103] (ab32499)

      Overlay histogram showing Jurkat cells stained with ab32499 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32499, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was anti-rabbit DyLight® 488 (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. 

       

       

    • Western blot - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
      Western blot - Anti-pro Caspase-3 antibody [E83-103] (ab32499)
      All lanes : Anti-pro Caspase-3 antibody [E83-103] (ab32499) at 1/10000 dilution

      Lane 1 : Jurkat cell lysate
      Lane 2 : Jurkat cell lysate + Camptothecin

      Predicted band size: 31 kDa
      Observed band size: 35 kDa why is the actual band size different from the predicted?

    • Anti-pro Caspase-3 antibody [E83-103] (ab32499)
      Anti-pro Caspase-3 antibody [E83-103] (ab32499)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (66)

    Publishing research using ab32499? Please let us know so that we can cite the reference in this datasheet.

    ab32499 has been referenced in 66 publications.

    • Zhang H  et al. KCNQ1OT1 regulates the retinoblastoma cell proliferation, migration and SIRT1/JNK signaling pathway by targeting miR-124/SP1 axis. Biosci Rep 41:N/A (2021). PubMed: 33345272
    • Zhang Z  et al. Role of lncRNA PART1 in intervertebral disc degeneration and associated underlying mechanism. Exp Ther Med 21:131 (2021). PubMed: 33376513
    • Wu Y  et al. Tetrandrine attenuates left ventricular dysfunction in rats with myocardial infarction. Exp Ther Med 21:119 (2021). PubMed: 33335582
    • Wang D  et al. Long non-coding RNA SNHG20 promotes ovarian cancer development by targeting microRNA-338-3p to regulate MCL1 expression. Oncol Lett 21:130 (2021). PubMed: 33552251
    • Luo Y  et al. Gypenoside XVII alleviates early diabetic retinopathy by regulating Müller cell apoptosis and autophagy in db/db mice. Eur J Pharmacol 895:173893 (2021). PubMed: 33493483
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    Question

    Which amino sequence does anti-cleaved caspase 3 recognize?

    One RabMAb primary antibody (ab32499) only detects pro-form of Caspase 3 and does not recognize any cleaved caspases, while the other (ab32042) only detects the active, cleaved form of Caspase 3 and does not recognize the pro-form.

    Can you please clarify the C3 amino acid sequence C8 cleaves (please specify between which two amino acids is the cut). And please specify which side anti-cleaved caspase 3 recognizes.

    Read More

    Abcam community

    Verified customer

    Asked on Dec 11 2014

    Answer

    Pro-caspase 3 is cleaved between amino acids 175 and 176 of the sequence described in the UniProt entry, P42574. http://www.uniprot.org/uniprot/P42574
    Antibody ab32042 is raised against the N terminus of subunit p17.

    Read More

    Tom Ruyle

    Abcam Scientific Support

    Answered on Dec 11 2014

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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