Caspase-3 Substrate DEVD-AFC

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Enzyme Activity Assay (EAA), Functional Studies (Func)
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Sequence Ac-Asp-Glu-Val-Asp-AFC
Characteristics AFC, 7-amino-4-trifluoromethyl coumarin
Purity > 98 % by HPLC
Chemical Name Ac-DEVD-AFC, Caspase-3 Substrate Fluorogenic
Formula C₃₀H₃₃F₃N₅O₁₃
Permeability Not-permeable
Molecular Weight 729 g/mol

Ready-to-use fluorometric substrate for caspase-3/CPP32 (Km = 9.7 µM) and related caspases that recognize the amino acid sequence DEVD. The sequence DEVD is based on caspase-3 cleavage site in poly (ADP-ribose) polymerase (PARP). CPP32 and related caspase activity can be quantified by fluorescent detection of free AFC after cleaved from the peptide substrate DEVD-AFC at Ex. = 400 nm and Em. = 505 nm, using a fluorometer or a 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.

Protocol 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 containing 10 mM DTT to each sample.
6. Add 5 µL of the 1 mM DEVD-AFC (50 µ M 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 caspase-3 activity can be determined by comparing these results with the level of the uninduced control.
Restrictions For Research Use only
Format Liquid
Handling Advice Potect from light and moisture
Storage -20 °C
Expiry Date 6-12 months
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