For optimal storage temperature of single components please refer to individual label. Avoid freeze/thaw cycles.
Shipped on Blue Ice
Short Term Storage:
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5x106 cells.
3. Resuspend cells in 50 µl of chilled Cell Lysis Buffer and incubate cells on ice for 10 min. Centrifuge for 1 min in a microcentrifuge (10’000 x g).
4. Transfer supernatant to a fresh tube and assay protein concentration.
5. Dilute 100-300 µg protein to 50 µl Cell Lysis Buffer for each test.
6. Add 50 µl of 2x Reaction Buffer containing 10 mM DTT to each sample.
7. Add 5 µl of the 4 mM pNA conjugated substrates (200 µM final conc.) into each tube individually and incubate at 37°C for 1-2 hours.
8. Read samples at 400 or 405nm in a microtiter plate reader, or spectrophotometer using a 100 µl micro quartz cuvet (Sigma), or dilute sample to 1ml with Dilution Buffer and using regular cuvet (note: dilution of the samples proportionally decreases the reading).
Fold-increase in caspase activity can be determined by comparing these results with the level of the uninduced control.
Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in caspase activity.