What does caspase-3 assay measure?
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.
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).
Is caspase-3 an enzyme?
Caspase 3 is a lysosomal enzyme involved in the apoptotic pathway, and is more specific in the detection of apoptotic cells than TUNEL (Figure 59.18).
What proteins does caspase-3 cleave?
Once activated, caspase-3 will cleave key structural proteins, cell cycle proteins, and DNase proteins, such as poly(ADP-ribose) polymerase, gelsolin, ICAD/DFF, and DNA-dependent kinase11,12,13. These cleavage events result in the blebbing and condensing of cells that ultimately leads to cell death14.
What is a caspase assay used for?
The FCC assay enables monitoring of cellular immune responses in real time.
How is caspase activity measured?
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 does caspase-3 indicate?
Thu, 04/07/2016 – 13:44. Caspases, or cysteine-dependent aspartate specific proteases, are a family of enzymes crucial for initiating and executing apoptosis within a cell, an important biological event especially during organ development (1).
How does cleaved caspase-3 work?
As an executioner caspase, the caspase-3 zymogen has virtually no activity until it is cleaved by an initiator caspase after apoptotic signaling events have occurred. One such signaling event is the introduction of granzyme B, which can activate initiator caspases, into cells targeted for apoptosis by killer T cells.
What is activated caspase-3?
Activation. Caspase-3 is activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways. The zymogen feature of caspase-3 is necessary because if unregulated, caspase activity would kill cells indiscriminately.
What is the molecular weight of caspase-3?
17kD
The processed form of Caspase 3 consists of large (theoretical molecular weight 17kD) and small (theoretical molecular weight 12kD) subunits which associate to form an active enzyme.
How is caspase-3 cleaved?
Caspase-3 is cleaved at an aspartate residue to yield a p12 and a p17 subunit to form the active caspase-3 enzyme [13]. Active caspase-3 degrades multiple cellular proteins and is responsible for morphological changes and DNA fragmentation in cells during apoptosis [9].
What is being detected in this assay to measure caspase activity?
The flow cytometry caspase (FCC) assay (Chahroudi et al., 2003; Jayaraman, 2003; Jerome et al., 2003; Telford et al., 2004) detects activation of caspase enzymes within target cells. This assay reveals the processes responsible for target-cell killing.
How does the assay detect activated caspase 3?
The assay is based on spectrophotometric detection of thechromophore p-nitroaniline (p-NA) after cleavage from the labeledsubstrate DEVD-p-NA. Therefore it should only recognize activated Caspase 3.
What are the subunits of caspase 3?
Caspase 3 is a relatively small protein that consists of 2 subunits, a 12- and 17-kDa subunit that contains 3 and 5 thiol functions, respectively.
Why is caspase-3 restricted to cells expressing Cre recombinase?
Caspase-3 is an executioner caspase, and leaky expression of the enzyme even at low levels can elicit apoptosis. We therefore wished to restrict caspase-3 expression exclusively to cells expressing Cre recombinase.
Why is caspase-3 such a good target for tethering?
Caspase-3, a cysteine-aspartyl protease that is one of the central ‘executioners’ of apoptosis, is an ideal target for tethering since the active-site cysteine residue can be readily covalently modified. The ‘extender’ strategy was chosen for the discovery of highly potent inhibitors ( Figure 9 ).