Product name : Caspase_8 Substrate IETD_AFC
Catalog number : 1062-200
Quantity: 200 assays
Supplier name : Biovis
Data sheet : Ask more or other datasheet now !
STORAGE: Store at –20°C, protected from light.
SHELF LIFE: 1 year under proper storage conditions
CONCENTRATION: 2 mM in DMSO
MOL. WEIGHT: 729.0
SEQUENCE: Ac-Ile-Glu-Thr-Asp-AFC (AFC, 7-amino-4-trifluoromethyl coumarin)
PURITY: >95% by HPLC analysis.
Ready-to-use fluorometric substrate for FLICE/caspase-8 and related caspases that recognize the amino acid sequence IETD. The sequence IETD is based on caspase-8 cleavage site in CPP32/caspase-3 proenzyme. FLICE and related caspase activity can be quantified by fluorescent detection of free AFC after cleavage from the peptide substrate IETD-AFC at Ex. = 400 nm and Em. = 505 nm, using a fluorometer or multi-well fluorescence plate reader. Alternatively, a shift in fluorescence from blue to green upon cleavage can be visualized using a hand-held long-UV lamp.
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5 x 106 cells or use 50-200 μg cell lysates if protein concentration has been measured.
3. Resuspend cells in 50 μl of chilled Cell Lysis Buffer (Cat.# 1067-100).
4. Incubate cells on ice for 10 minutes.
5. Add 50 μl of 2X Reaction Buffer (Cat.# 1068-20, -80) containing 10 mM DTT (Cat.#1201-1) to each sample.
6. Add 5 μl of the 2 mM IETD-AFC (100 μM final conc.) into each tube individually and incubate at 37°C for 1-2 hour.
7. Read samples in a fluorometer equipped with a 400-nm excitation filter and 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.
8. Fold-increase in IETD-dependent caspase-8 activity can be determined by comparing these results with the level of the uninduced control.
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