Key features and details
- Assay type: Enzyme activity
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell Lysate, Tissue Lysate
Product nameCaspase-3 Assay Kit (Colorimetric)
See all Caspase-3 kits
Sample typeCell Lysate, Tissue Lysate
Assay typeEnzyme activity
Assay time2h 00m
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
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
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
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-pNA 1 x 500µl Dilution Buffer 1 x 100ml 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
- CASP 3
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).
ab39401 has been referenced in 141 publications.
- Wu Z et al. Dioscin Inhibited Glycolysis and Induced Cell Apoptosis in Colorectal Cancer via Promoting c-myc Ubiquitination and Subsequent Hexokinase-2 Suppression. Onco Targets Ther 13:31-44 (2020). PubMed: 32021252
- Shimizu Y et al. Role of DJ-1 in Modulating Glycative Stress in Heart Failure. J Am Heart Assoc 9:e014691 (2020). PubMed: 32067589
- Hwang SK et al. PDCD4 inhibits lung tumorigenesis by the suppressing p62-Nrf2 signaling pathway and upregulating Keap1 expression. Am J Cancer Res 10:424-439 (2020). PubMed: 32195018
- Zhang HY et al. Prenatal low-dose endotoxin exposure prolongs intestinal epithelial activation after birth and contributes to necrotizing enterocolitis. J Pediatr Surg N/A:N/A (2020). PubMed: 32321629
- Zhao L et al. YY1-mediated PTEN dephosphorylation antagonizes IR-induced DNA repair contributing to tongue squamous cell carcinoma radiosensitization. Mol Cell Probes N/A:101577 (2020). PubMed: 32334006