Caspase-3 Fluorometric Assay Kit
Caspase-3 Assay Kit (Fluorometric)
Detection method: Fluorescent
Tissue extracts, cell lysate
Test type: Enzymatic activity
Test time: 2h 00m
The Caspase-3 (Fluorometric) Assay Kit (ab39383) provides a simple and convenient means of testing DVD-dependent caspase activity. The Caspase-3 assay protocol is based on the detection of cleavage of the substrate DVD-AFC (AFC: 7-amino-4-trifluoromethylcoumarin). DVD-AFC emits blue light (λ max = 400 nm).
On cleavage of the substrate by CPP32 or related caspases, free CFA emits a greenish-yellow fluorescence (Ex / Em = 400/505 nm). The signal can be quantified using a fluorometer or fluorescence microtiter plate reader. Comparison of the CFA fluorescence of an apoptotic sample with an uninduced control allows the 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 DVD-AFC substrate and incubate for 1-2 hours at 37ºC
- analyze with fluorometer or microplate reader
Due to the nature of the substrate, this assay also detects caspase-7 activity.
Other caspase and apoptosis assays
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Platform: Microplate reader
Store at -20 ° C. See protocols.
Involved in the caspase activation cascade responsible for the execution of apoptosis. At the onset of apoptosis, it proteolytically cleaves poly (ADP-ribose) polymerase (PARP) at a ‘216-Asp-Gly-217 ‘link. It cleaves and activates the sterol regulatory element-binding proteins (SREBPs) between the basic helix-loop-helix leucine zipper domain and the membrane-binding domain. Cleavage and activate caspase-6, -7, and -9. Involved in huntingtin cleavage.
Highly expressed in the lung, spleen, heart, liver, and kidney. Moderate levels in the brain and skeletal muscle and low in testes. It is also found in many cell lines, the highest expression in the cells of the immune system.
It belongs to the C14A peptidase family.
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 also appear between the small subunit of caspase-7 protease and the large subunit of caspase-3 and vice versa. S-nitrosylated at its catalytic site cysteine in unstimulated human cell lines and denitrosylated after activation of the apoptotic Fas pathway, associated with an increase in intracellular caspase activity. Thus, Fas activates caspase-3 not only by inducing caspase zymogen cleavage into its active subunits but also by stimulating denitrosylation of its thiol active site.
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