The Caspase 8 quantification kit is a bioluminescent assay that measures caspase 8 activities in vitro, as well as in vivo using the split-luciferin technology. Caspase 8 is cysteine/aspartic acid-specific protease and play key effector roles in apoptosis in mammalian cells1–2.
The split luciferin approach enables the generation of D-luciferin through the reaction of D-Cysteine and 6-amino-2-cyanobenzothiazole (NH2-CBT). The D-Cysteine amino acid has been caged with a caspase 8 specific peptidic IETD sequence. The modification of the amino group on D-Cysteine prohibits reaction with the counterpart NH2-CBT since the 1,2-aminothiol is required for the reaction with the cyano group. After cleavage of the peptide by Caspase 8 free D-Cysteine is liberated and can further react with NH2-CBT to form Luciferin and then bioluminescence through oxydation by Luciferase (Figure 1). Luminescence is thus proportional to the amount of active caspase 8.
One of the advantages of the split luciferin approach is improved cell penetration property of CBT derivatives in comparison to full luciferin scaffold. The Caspase 8 quantification kit is also more sensitive than fluorescence-based caspase assays. The assay uses a substrate containing the IETD sequence that is selective for caspase 8.3-4
This technology has been developed to measure caspase 8 acitivity in live cells but also in living animals. The caspase 8 quantification kit provide a very high signal to background ratio allowing sensitive detection of caspase 8 activity.
The assay provides a luminogenic caspase-8 substrate, which contains the tetrapeptide sequence linked to D-Cysteine Z-Ile-Glu-Thr-Asp-D-Cys (Z-IETD-(D-Cys)), and 6-amino-2-cyanobenzothiazole (NH2-CBT).