Central to the execution phase of apoptosis are the two closely related caspase-3 and caspase-7. They share common substrate specificity and structure, but differ completely in the sequence of their respective N-terminal regions. Both caspase-3 and caspase-7 have substrate selectivity for the amino acid sequence Asp-Glu-Val-Asp (DEVD). The SensoLyte® Homogeneous Rh110 Capase-3/7 Assay Kit uses (Ac-DEVD)2-Rh110 as the fluorogenic indicator for assaying caspase-3/7 activities. Upon caspase-3/7 cleavage, (Ac-DEVD)2-Rh110 generates the Rh110 fluorophore which has bright green fluorescence and can be detected at Ex/Em=496 nm/520 nm. The longer-wavelength spectra and higher extinction coefficient of the Rh110 provide greater sensitivity and less interference from cell components.A bi-function assay buffer in this kit is designed to lyze the cells and measure the enzyme activity at the same time. Thus, this kit can measure caspase-3/7 activity in cell culture directly in a 96-well or 384-well plate without a time-consuming cell extraction step. In case the cells are cultured in larger plates or flasks, a lysis buffer and protocol for cell lysate preparation are also conveniently included in the kit. The kit is suitable for high throughput screening of apoptosis inducers and inhibitors.
Component A: Rh110 Caspase-3/7 substrate Peptide sequence=(Z-Asp-Glu-Val-Asp)2-Rh110, Ex/Em=496 nm/520 nm upon caspase-3/7 cleavage: 250 µL
Component B: Rh110, fluorescence reference standard, Ex/Em=496 nm/520 nm: 1 mM DMSO solution, 40 µL
Component C: Ac-DEVD-CHO, a known caspase-3/7 inhibitor Peptide sequence=Ac-Asp-Glu-Val-Asp-CHO: 5 mM DMSO solution, 10 µL
Component D: Assay buffe:r 30 mL
Component E: DTT: 1 M, 1.1 mL
Component F: 10X Lysis Buffer: 20 mL
Storage & stability
Store components A, B, C, and E at -20°C. Components D and F can be stored at 4°C for convenience. Component A and B protect from light and moisture.