Caspase-8 Substrate IETD-AFC

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Application
Enzyme Activity Assay (EAA), Functional Studies (Func)
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Sequence Ac-Ile-Glu-Thr-Asp-AFC
Purity > 95 % by HPLC
Chemical Name Ac-IETD-AFC, Caspase-8 Substrate, Fluorogenic
Formula C₃₁H₃₈F₃N₅O₁₂
Permeability Not-permeable
Molecular Weight 729.7 g/mol
Comment

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.

Protocol 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5 x 10^6 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 IETD-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. Fold-increase in IETD-dependent caspase-8 activity can be determined by comparing these results with the level of the uninduced control.
Restrictions For Research Use only
Format Liquid
Handling Advice Protect from light and moisture
Storage -20 °C
Expiry Date 12 months
Product cited in: Bok, Prikhodko, Green, Sosnovtsev: "Apoptosis in murine norovirus-infected RAW264.7 cells is associated with downregulation of survivin." in: Journal of virology, Vol. 83, Issue 8, pp. 3647-56, 2009 (PubMed).

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Ghosh, Pulinilkunnil, Yuen, Kewalramani, An, Qi, Abrahani, Rodrigues: "Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion." in: American journal of physiology. Heart and circulatory physiology, Vol. 289, Issue 2, pp. H768-76, 2005 (PubMed).

Ray, Akbiyik, Bernstein, Phipps: "CD40 engagement prevents peroxisome proliferator-activated receptor gamma agonist-induced apoptosis of B lymphocytes and B lymphoma cells by an NF-kappaB-dependent mechanism." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 174, Issue 7, pp. 4060-9, 2005 (PubMed).

Wesche-Soldato, Chung, Lomas-Neira, Doughty, Gregory, Ayala: "In vivo delivery of caspase-8 or Fas siRNA improves the survival of septic mice." in: Blood, Vol. 106, Issue 7, pp. 2295-301, 2005 (PubMed).

Cummings, McHowat, Schnellmann: "Role of an endoplasmic reticulum Ca2+-independent phospholipase A2 in cisplatin-induced renal cell apoptosis." in: The Journal of pharmacology and experimental therapeutics, Vol. 308, Issue 3, pp. 921-8, 2004 (PubMed).

Keedwell, Zhao, Hammond, Qin, Tsang, Reitmair, Molina, Okawa, Atangan, Shurland, Wen, Wallace, Bird, Chandraratna, Brown: "A retinoid-related molecule that does not bind to classical retinoid receptors potently induces apoptosis in human prostate cancer cells through rapid caspase activation." in: Cancer research, Vol. 64, Issue 9, pp. 3302-12, 2004 (PubMed).

Li, Perlman, Peterson, Burrichter, Avdulov, Polunovsky, Bitterman: "Translation initiation factor 4E blocks endoplasmic reticulum-mediated apoptosis." in: The Journal of biological chemistry, Vol. 279, Issue 20, pp. 21312-7, 2004 (PubMed).

Bai, Goodrich: "Different DNA lesions trigger distinct cell death responses in HCT116 colon carcinoma cells." in: Molecular cancer therapeutics, Vol. 3, Issue 5, pp. 613-9, 2004 (PubMed).

Nowak, Price, Schnellmann: "Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells." in: American journal of physiology. Renal physiology, Vol. 285, Issue 3, pp. F440-50, 2003 (PubMed).

Coletti, Yang, Marazzi, Sassoon: "TNFalpha inhibits skeletal myogenesis through a PW1-dependent pathway by recruitment of caspase pathways." in: The EMBO journal, Vol. 21, Issue 4, pp. 631-42, 2002 (PubMed).

Cummings, Schnellmann: "Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways." in: The Journal of pharmacology and experimental therapeutics, Vol. 302, Issue 1, pp. 8-17, 2002 (PubMed).

Daniel, Duffield, Brunner, Steinmann-Niggli, Lods, Marti: "Matrix metalloproteinase inhibitors cause cell cycle arrest and apoptosis in glomerular mesangial cells." in: The Journal of pharmacology and experimental therapeutics, Vol. 297, Issue 1, pp. 57-68, 2001 (PubMed).