Sample Type:Cell lysates and fresh tissue homogenate
Intended Use:Caspase-3 Assay Kit provides the reagents needed for a quick and efficient detection of Caspase-3 activity in cell lysates and fresh tissue homogenate.
Test Principle:Caspases (Cysteine-requiring Aspartate protease) are a family of proteases that mediate cell death and are important to the process of apoptosis. Caspase-3 (also referred to as CPP32, Yama, and apopain) is a member of the CED-3 subfamily of caspases and is one of the critical enzymes of apoptosis. Caspase-3, which is an effector caspase, is the most studied of mammalian caspases. Caspase-3, which is an effector caspase, is the most studied of mammalian caspases. It can process procaspase 2, 6, 7, and 9 and specifically cleave most of caspase related substrates known to date, including many key proteins such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP), the inhibitor of caspase-activated deoxyribonuclease (ICAD), and gelsolin and fodrin, which are proteins involved in apoptosis regulation. This cleavage is part of the mechanism leading to cell death. In addition, caspase-3 plays a central role in mediating nuclear apoptosis including chromatin condensation and DNA fragmentation as well as cell blebbing. Caspase-3 activity is tissue, cell type, or death stimulus specific. The caspase-3 assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac- DEVD-pNA) by caspase-3, resulting in the release of the p-nitroaniline (pNA) moiety. p-Nitroaniline has a high absorbance at 405nm. The concentration of the pNA released from the substrate is calculated from the absorbance values at 405nm or from a calibration curve prepared with defined pNA solutions.
Kit Components (see Protocol for final kit quantities):Lysis Buffer, 10.0mLReaction Bufferx2, 2.5mLAc-DEVD-pNA, 250uLDTT, 100uL
Storage and Stability:Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
