Overview

  • Product name
    Caspase-3 Assay Kit (Colorimetric)
    See all Caspase-3 kits
  • Detection method
    Colorimetric
  • Sample type
    Cell Lysate
  • Assay type
    Enzyme activity
  • Assay time
    2h 00m
  • Product overview

    Caspase-3 Assay Kit (Colorimetric) ab39401 provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD.


    The Caspase-3 assay protocol is based on the formation of the chromophore p-nitroaniline (p-NA) by cleavage from the labeled substrate DEVD-pNA. The p-NA can be quantified using a spectrophotometer or a microtiter plate reader reading absorbance at 400 or 405 nm.


    Comparison of the absorbance of p-NA from an apoptotic sample with an uninduced control allows determination of the fold increase in Caspase-3 activity.


    Caspase-3 assay protocol summary:
    - add samples to wells
    - add reaction buffer and DEVD-p-NA substrate and incubate for 60-120 min at 37ºC
    - analyze with microplate reader

  • Notes

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

    Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells.

    Other caspase and apoptosis assays

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

  • Platform
    Microplate reader

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-pNA 1 x 500µl
    Dilution Buffer 1 x 100ml
    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

  • H9c2 cells were either untreated (control) or treated with 2µg/ml of recombinant lipocalin-2 (Lcn2) for 24h. Cell lysates were assayed for caspase-3 activity (n = 3, *, p<0.05).

    Image obtained from Xu G et al., JBC, 2012 Feb 11;28(7):4808-17

  • Caspase-3 in Jurkat lysates (3.3 x10e6 cells) following 20 hour exposure to 2 uM Camptothecin (ab120115) or 10 ng/mL anti-Fas Ab (MBL).

Protocols

References

This product has been referenced in:
  • Ponder KG & Boise LH The prodomain of caspase-3 regulates its own removal and caspase activation. Cell Death Discov 5:56 (2019). Read more (PubMed: 30701088) »
  • Li Y  et al. HOXB4 knockdown enhances the cytotoxic effect of paclitaxel and cisplatin by downregulating ABC transporters in ovarian cancer cells. Gene 663:9-16 (2018). Read more (PubMed: 29660518) »
See all 64 Publications for this product

Customer reviews and Q&As

Filter by Ratings

It doesn't work for tissue samples

Inconclusive Good 4/5 (Ease of Use)
Abreviews
We tried to quantify Caspase 3 activity in mouse mammary gland during mammary involution after lactation. We tested different protein amounts fron 50 to 200 microgrames (as indicated in protocol booklet), but the product did not work in any case. We wanted to compare Caspase 3 activity in three samples at different timepoints of involution, and we were not able to observe differences (A), which really exist, as we confirmed by western blot of the same samples (B). We checked the kit was not faulty testig in HC11 mammary cell line, inducing apoptosis with cisplatin 15 ug/ml for 24h (C).
The technical service told us it was a problem with our samples and the did not offer a way to solve our problem.
Then, we found a fluorimetric assay ab39383, which is exactly the same but with a fluorometric substrate instead of a colorimetric one, which is more sensitive as abcam scientific support says in the questions and answer section of the product wall in their website. We aquired the fluorometric substrate and repeated the assay with the same samples. This time, the product worked perfectly (D).
So, for mouse mammary gland tissue samples, I would not recommend this product. It seems it has not enough sensitivity. I would try the fluorimetric assay.

Mr. Adrian Blanco

Verified customer

Submitted Jul 17 2014

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