Caspase-8 Substrate IETD-AFC

Application: Functional Studies (Func), Enzyme Activity Assay (EAA)

Catalog no.: ABIN412373

Additional Information

Immunogen: Sequence: Ac-Ile-Glu-Thr-Asp-AFC (AFC, 7-amino-4-trifluoromethyl coumarin)

Description: 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.

Molecular Weight: 729.0

Application Details

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 μ g cell lysates if protein concentration has been measured. 3. Resuspend cells in 50 μ l of chilled Cell Lysis Buffer (Cat.# 1067-100). 4. Incubate cells on ice for 10 minutes. 5. Add 50 μ l of 2X Reaction Buffer (Cat.# 1068-20, -80) containing 10 mM DTT (Cat.# 1201-1) to each sample. 6. Add 5 μ l of the 1 mM IETD-AFC (50 μ M final conc.) into each tube individually and incubate at 37 o 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.

Purity: >95% by HPLC analysis.

Restrictions: For Research Use only

Publications

Product: Daniel, Duffield, Brunner et al.: "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).

Coletti, Yang, Marazzi et al.: "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).

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).

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

Keedwell, Zhao, Hammond et al.: "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).

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).

Ray, Akbiyik, Bernstein et al.: "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).

Ghosh, Pulinilkunnil, Yuen et al.: "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).

Wesche-Soldato, Chung, Lomas-Neira et al.: "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).

Chanvorachote, Nimmannit, Wang et al.: "Nitric oxide negatively regulates Fas CD95-induced apoptosis through inhibition of ubiquitin-proteasome-mediated degradation of FLICE inhibitory protein." in: The Journal of biological chemistry, Vol. 280, Issue 51, pp. 42044-50, 2005 (PubMed).

Bok, Prikhodko, Green et al.: "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).