Caspase 3 Colorimetric Substrate (N-acetyl-Asp-Glu-Val-Asp-pNA (p-nitroanilide)) (CPP-32, Apoptain, Yama, SCA-1)

Colorimetric substrate for Caspase-3. Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. See Caspase-3 Apoptosis Detection Kit C2087-20 which provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD. The assay is based on detection of cleavage of substrate DEVD-pNA (p-nitroanilide). Upon proteolytic cleavage of the substrate by caspases, free pNA is detected at a wavelength of 400nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the increase in Caspase-3 activity. Sequence: Ac-Asp-Glu-Val-Asp-pNA Storage and Stability: May be stored at 4 degrees C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. Protocol: 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. Count cells and pellet 1-5x10e6 cells. 3. Resuspend cells in 50ul of chilled Cell Lysis Buffer (C2086-10F) and incubate cells on ice for 10 minutes. 4. Centrifuge for 1 min (10,000 x g). 5. Transfer supernatant (cytosolic extract) to a fresh tube and put on ice. 6. Assay protein concentration. 7. Dilute 50-200ug protein to 50ul Cell Lysis Buffer for each assay. 8. Add 50ul of 2X Reaction Buffer (C2086-10F1) containing 10 mM DTT to each sample. 9. Add 5ul of the 4mM C2087-21 (200uM final conc.) and incubate at 37C for 1-2 hours. 9. Read samples at 400 or 405nm in a microtiter plate reader or spectrophotometer using a 100ul micro quartz cuvet, or dilute sample to 1ml with Dilution Buffer using a regular cuvet.