Product nameCaspase 3 Assay Kit (Fluorometric)
See all Caspase-3 kits
Sample typeTissue Extracts, Cell Lysate
Assay typeEnzyme activity
Assay time2h 00m
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.
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Activation of ICE-family proteases/caspases initiates apoptosis or other cellular processes in mammalian cells.
Storage instructionsStore 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
FunctionInvolved 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 specificityHighly 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 similaritiesBelongs to the peptidase C14A family.
modificationsCleavage 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.
- Information by UniProt
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:
- 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) »