Caspase-3 Substrate DEVD-AFC

Details for Product No. ABIN412341
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Application
Functional Studies (Func), Enzyme Activity Assay (EAA)
Pubmed 19 references available
Quantity 1000 tests
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Catalog No. ABIN412341
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Immunogen AFC, 7-amino-4-trifluoromethyl coumarin
Sequence Ac-Asp-Glu-Val-Asp-AFC
Purity > 98 % by HPLC
Molecular Weight 729 Da
Comment

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
Product cited in: Hall, Matter, Wang et al.: "Hyperglycemia inhibits vascular smooth muscle cell apoptosis through a protein kinase C-dependent pathway." in: Circulation research, Vol. 87, Issue 7, pp. 574-80, 2000 (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).

Nowak: "Protein kinase C-alpha and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells." in: The Journal of biological chemistry, Vol. 277, Issue 45, pp. 43377-88, 2002 (PubMed).

Sherwood, Sternberg: "Anchor cell invasion into the vulval epithelium in C. elegans." in: Developmental cell, Vol. 5, Issue 1, pp. 21-31, 2003 (PubMed).

Suh, Mutoh, Nagashima et al.: "The organellular chloride channel protein CLIC4/mtCLIC translocates to the nucleus in response to cellular stress and accelerates apoptosis." in: The Journal of biological chemistry, Vol. 279, Issue 6, pp. 4632-41, 2004 (PubMed).

Jilling, Lu, Jackson et al.: "Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental rat model of neonatal necrotizing enterocolitis." in: Pediatric research, Vol. 55, Issue 4, pp. 622-9, 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).

Yano, Itoh, Kubota et al.: "A prostacyclin analog prevents radiocontrast nephropathy via phosphorylation of cyclic AMP response element binding protein." in: The American journal of pathology, Vol. 166, Issue 5, pp. 1333-42, 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).

Feng, Bianchi, Sandmeyer et al.: "Bradykinin preconditioning improves the profile of cell survival proteins and limits apoptosis after cardioplegic arrest." in: Circulation, Vol. 112, Issue 9 Suppl, pp. I190-5, 2005 (PubMed).

Mohammad, Goustin, Aboukameel et al.: "Preclinical studies of TW-37, a new nonpeptidic small-molecule inhibitor of Bcl-2, in diffuse large cell lymphoma xenograft model reveal drug action on both Bcl-2 and Mcl-1." in: Clinical cancer research : an official journal of the American Association for Cancer Research, Vol. 13, Issue 7, pp. 2226-35, 2007 (PubMed).

Claud, Lu, Wang et al.: "Platelet-activating factor-induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells." in: American journal of physiology. Gastrointestinal and liver physiology, Vol. 294, Issue 5, pp. G1191-200, 2008 (PubMed).

Lu, Caplan, Li et al.: "Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells." in: American journal of physiology. Gastrointestinal and liver physiology, Vol. 294, Issue 5, pp. G1181-90, 2008 (PubMed).

Griswold, Chang, Runko et al.: "Sir2 mediates apoptosis through JNK-dependent pathways in Drosophila." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, Issue 25, pp. 8673-8, 2008 (PubMed).

Sémiramoth, Gleizes, Turbica et al.: "Afa/Dr-expressing, diffusely adhering Escherichia coli strain C1845 triggers F1845 fimbria-dependent phosphatidylserine externalization on neutrophil-like differentiated PLB-985 cells through an apoptosis-independent mechanism." in: Infection and immunity, Vol. 78, Issue 7, pp. 2974-83, 2010 (PubMed).

Rui, Zhang, Xu et al.: "Reduction in IL-33 expression exaggerates ischaemia/reperfusion-induced myocardial injury in mice with diabetes mellitus." in: Cardiovascular research, Vol. 94, Issue 2, pp. 370-8, 2012 (PubMed).

Ren, Lu, Porollo et al.: "Tumor-suppressing function of caspase-2 requires catalytic site Cys-320 and site Ser-139 in mice." in: The Journal of biological chemistry, Vol. 287, Issue 18, pp. 14792-802, 2012 (PubMed).

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