Fluorometric substrates for assaying activities of members of caspase family proteases. All substrates are provided in liquid ready-to-use form.
|Kit/Set Contains:||Contains 125µl (1mM) each of the following substrates:|
- Caspase-1 Substrate, Ac-YVAD-AFC
- Caspase-2 Substrate, Ac-VDVAD-AFC
- Caspase-3 Substrate, Ac-DEVD-AFC
- Caspase-5 Substrate, Ac-WEHD-AFC
- Caspase-6 Substrate, Ac-VEID-AFC
- Caspase-8 Substrate, Ac-IETD-AFC
- Caspase-9 Substrate, Ac-LEHD-AFC
|Formulation:||Liquid. Ready-to-use in DMSO.|
|Shipping:||Shipped on Blue Ice|
|Long Term Storage:||-20°C|
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5x106 cells or use 50-200µg cell lysates if protein concentration has been measured.
3. Resuspend cells in 50µl of chilled Cell Lysis Buffer.
4. Incubate cells on ice for 10 minutes.
5. Add 50µl of 2x Reaction Buffer containing 10mM DTT to each sample.
6. Add 5µl of the 1mM AFC conjugated substrates (50µM final conc.) into each tube individually and incubate at 37°C for 1-2 hours.
7. Read samples in a fluorometer equipped with a 400-nm excitation filter and a 505-nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. You may perform the entire assay directly in a 96 well plate.
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.