• Product name
    Caspase-3 Assay Kit (Fluorometric)
    See all Caspase-3 kits
  • Detection method
  • 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 DEVD-dependent caspase activity.

    The Caspase-3 assay protocol 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). On cleavage of the substrate by CPP32 or related caspases, free AFC emits a yellow-green fluorescence (Ex/Em = 400/505 nm).

    The signal 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.

    Caspase-3 assay protocol summary:
    - lyse cells / homogenize and lyse tissues in lysis buffer
    - incubate on ice for 10 min
    - add reaction buffer and DEVD-AFC substrate and incubate for 1-2 hr at 37ºC
    - analyze with fluorometer or microplate reader


  • Notes

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

    Other caspase and apoptosis assays

    Review the full set of caspase assays, or the apoptosis assay and apoptosis marker guide.

  • Platform
    Microplate reader


  • 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
    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
  • Information by UniProt
  • Alternative names
    • A830040C14Rik
    • Apopain
    • CASP-3
    • CASP3
    • 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
    • LICE
    • mldy
    • OTTHUMP00000165052
    • OTTHUMP00000165053
    • OTTHUMP00000165054
    • PARP cleavage protease
    • Procaspase3
    • Protein Yama
    • SCA 1
    • SCA-1
    • SREBP cleavage activity 1
    • Yama
    see all


  • 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.

  • 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.



This product has been referenced in:
See all 16 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A


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-AFC by Caspase-3. Inhibition of caspase would thus interfere with the measurement.

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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.    

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Unfortunately, this is proprietary information.

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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 can detect Caspase 3 in almost all mammalian cells. I hope this information will be useful for you.

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