Key features and details
- Assay type: Enzyme activity
- Detection method: Fluorescent
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell Lysate, Tissue Extracts
参阅全部 Caspase-3 试剂盒
样品类型Tissue Extracts, Cell Lysate
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
This product is manufactured by BioVision, an Abcam company and was previously called K105 Caspase-3 Fluorometric Assay Kit. K105-100 is the same size as the 100 test size of ab39383.
Due to the nature of the substrate, this assay also detects caspase-7 activity.
Other caspase and apoptosis assays
存放说明Store at -20°C. Please refer to protocols.
组件 100 tests 2X Reaction Buffer 4 x 2ml Cell Lysis Buffer 1 x 100ml DEVD AFC 1 x 500µl DTT 1 x 400µl
功能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.
组织特异性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.
序列相似性Belongs to the peptidase C14A family.
翻译后修饰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.
- Information by UniProt
- CASP 3
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.
ab39383 被引用在 92 文献中.
- Jiang Y et al. Cytoplasmic sequestration of p53 by lncRNA-CIRPILalleviates myocardial ischemia/reperfusion injury. Commun Biol 5:716 (2022). PubMed: 35851102
- Beillevaire D et al. Autolysosomes and caspase-3 control the biogenesis and release of immunogenic apoptotic exosomes. Cell Death Dis 13:145 (2022). PubMed: 35149669
- Fanfone D et al. Confined migration promotes cancer metastasis through resistance to anoikis and increased invasiveness. Elife 11:N/A (2022). PubMed: 35256052
- Homayoun M et al. In vitro evaluation of the pogostone effects on the expression of PTEN and DACT1 tumor suppressor genes, cell cycle, and apoptosis in ovarian cancer cell line. Res Pharm Sci 17:164-175 (2022). PubMed: 35280836
- Tomaszewska E et al. Prenatal acrylamide exposure results in time-dependent changes in liver function and basal hematological, and oxidative parameters in weaned Wistar rats. Sci Rep 12:14882 (2022). PubMed: 36050419