How is caspase-3 activity measured?
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.
What is caspase-3 activity assay?
Description: The Caspase-3 Activity Assay Kit is a fluorescent assay that detects the activity of caspase-3 in cell lysates. Cleavage of the substrate only occurs in lysates of apoptotic cells; therefore, the amount of AMC produced is proportional to the number of apoptotic cells in the sample.
Is cleaved caspase-3 active?
Caspase-3 is cleaved at an aspartate residue to yield a p12 and a p17 subunit to form the active caspase-3 enzyme [13]. In addition, cleaved caspase-3 promotes chemical- or radiation-induced DNA damage and oncogenesis [18].
What is a caspase assay used for?
The assay can be used to screen for caspase inhibitors as well as inducers. To screen for caspase inducers, lysates from cells treated with compounds at various doses and time points are analyzed. To screen for caspase inhibitors, either purified enzymes or treated cells can be analyzed.
How do you test caspase activity?
Caspase activity was determined by quantifying fluorescence images after excitation at 380 nm and emission centered at both 545 nm (yellow fluorescence, i.e. FRET) and 450 nm (blue fluorescence, i.e. conventional excitation) were captured.
What is the role of caspase 3 in apoptosis?
Caspase-3 is known as an executioner caspase in apoptosis because of its role in coordinating the destruction of cellular structures such as DNA fragmentation or degradation of cytoskeletal proteins (1).
How does caspase colorimetric assay work?
The caspase-3 colorimetric assays measure the proteolytic cleavage of the chromophore p-nitroanilide (pNA) by caspase-3. Liberated pNA can be monitored colorimetrically by absorbance at 405 nm. Fluorescence detection is highly sensitive and can be used to measure even very small amounts of active caspase.
What is a caspase assay?
Description. The Caspase-Glo® 3/7 Assay(a,b) is a homogeneous, luminescent assay that measures caspase-3 and -7 activities. The assay provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity and cell lysis.
Which is the colorimetric caspase 3 assay kit?
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.
How is the activity of caspase 3 determined?
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. Due to the nature of the substrate, this assay also detects caspase-7 activity.
How does FAS activate the caspase zymogen?
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. Cytoplasm. Nucleic Acids Res. 38:D142-D148 (2010) .