Overview

  • Product name
    Caspase 3 Assay Kit (Fluorometric)
    See all Caspase-3 kits
  • Sample type
    Tissue Extracts, Cell Lysate
  • Assay type
    Enzyme activity
  • Assay time
    2h 00m
  • Product overview

    Caspase 3 Assay Kit (Fluorometric) (ab39383) provides a simple and convenient means for assaying the DEVD-dependent caspase activity. The assay is based on detection of cleavage of substrate DEVD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). DEVD-AFC emits blue light (λ max = 400 nm); upon cleavage of the substrate by CPP32 or related caspases, free AFC emits a yellow-green fluorescence (Ex/Em = 400/505 nm), which can be quantified using a fluorometer or a fluorescence microtiter plate reader. Comparison of the fluorescence of AFC from an apoptotic sample with an uninduced control allows determination of the fold increase in caspase-3/CPP32 activity.


    Due to the nature of the substrate, this assay also detects caspase 7 activity.


    Visit our FAQs page for tips and troubleshooting.

  • Notes

    Activation of ICE-family proteases/caspases initiates apoptosis or other cellular processes in mammalian cells.

Properties

  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    2X Reaction Buffer 4 x 2ml
    Cell Lysis Buffer 1 x 100ml
    DEVD AFC 1 x 500µl
    DTT 1 x 400µl
  • Research areas
  • 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
  • Alternative names
    • A830040C14Rik
    • Apopain
    • CASP-3
    • CASP3
    • CASP3_HUMAN
    • Casp3a
    • Caspase 3
    • Caspase 3, apoptosis-related cysteine peptidase
    • Caspase 3, apoptosis-related cysteine protease
    • Caspase 3, apoptosis-related cysteine protease a
    • Caspase-3 subunit p12
    • CC3
    • CPP-32
    • CPP32
    • CPP32B
    • Cysteine protease CPP32
    • EC 3.4.22.56
    • LICE
    • mldy
    • OTTHUMP00000165052
    • OTTHUMP00000165053
    • OTTHUMP00000165054
    • PARP cleavage protease
    • Procaspase3
    • Protein Yama
    • SCA 1
    • SCA-1
    • SREBP cleavage activity 1
    • Yama
    see all

Images

  • Functional assays: Caspase 3 assay kit (ab39383)
    Functional assays: Caspase 3 assay kit (ab39383)Image from Lin S.F et al., PLoS One 12(2). Fig4B. doi: 10.1371/journal.pone.0172315.

    Caspase-3 activity was analyzed using fluorometric assay kit (ab39383). Thyroid cancer cells were plated at 1 x 106 cells in 10 mL of media and incubated  overnight. Cells were treated with Dinaciclib (25 nM) for 24 hours. Adherent cells (5 x 105) were collected, centrifuged, lysed using 50 μL of lysis buffer on ice for 10 min, incubated with DEVD-AFC substrate and reaction buffer at 37°C for 1.5 hours. Caspase-3 activity was detected by spectrophotometry. The fluorescence intensity of the treated samples was compared with that of control samples to determine the fold-increase in caspase activity. Each condition was performed in duplicate.

  • Functional assays: Caspase 3 Assay Kit (Fluorometric) (ab39383)
    Functional assays: Caspase 3 Assay Kit (Fluorometric) (ab39383)

    Caspase-3 activity in Jurkat lysates (6.6 x10e5 cells) following 20 hour exposure to 2 uM Camptothecin (ab120115) or 10 ng/mL anti-Fas Ab (MBL). Background signal subtracted, duplicates +/- SD.

Protocols

References

This product has been referenced in:
  • Zou ZZ  et al. Synergistic induction of apoptosis by salinomycin and gefitinib through lysosomal and mitochondrial dependent pathway overcomes gefitinib resistance in colorectal cancer. Oncotarget 8:22414-22432 (2017). Functional Studies ; Human . Read more (PubMed: 26461472) »
  • Tao CC  et al. Epigenetic regulation of HDAC1 SUMOylation as an endogenous neuroprotection against Aß toxicity in a mouse model of Alzheimer's disease. Cell Death Differ 24:597-614 (2017). Functional Studies . Read more (PubMed: 28186506) »

See all 10 Publications for this product

Publishing research using ab39383? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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4 reviews or Q&A

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I am interested in using Abcam Caspase 3 assay kit (ab39383) in samples from rat's heart. However, I already have the tissue lysate. Do the protease inhibitor that I added will interfere with the assay?



I am afraid that your protease inhibitors might interefere with the assay. Since Caspase is a cysteine protease it is probably inhibited by most protease inhibitor cocktails. The assay is based on the catalytic clevage of the substrate DEVD-A...

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Will this kit detect full length caspase 3 or cleaved caspase 3?

Thank you for your inquiry. This kit will detect active Caspase-3 from samples. The procasepase-3 (inactive) is proteolytically processed into the active Caspase-3. I hope this information helps.  Please contact us with any other questions.

Thank you for your reply. Our user wants know detail component of cell lysis buffer.

Unfortunately, this is proprietary information.

i need senstivity range and Species Reactivity for ab 39401 and AB 3983. i need these kit for rats.

Thank you for your enquiry and your interest in our products. The sensitivity range for the fluorometric substrate (K105) is in the tenths of a µM, while the sensitivity of the colorimetric substrates (K106) is in the single digit µM. These kits ca...

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