Caspase-9 Substrate LEHD-AFC

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

Catalog no.: ABIN412395

Additional Information

Immunogen: Sequence: Ac-Leu-Glu-His-Asp-AFC

Description: Ready-to-use fluorometric substrate for caspase-9/Mch-6 and related caspases that recognize the amino acid sequence LEHD. Caspase activity can be quantified by fluorescent detection of free AFC after cleavage from the peptide substrate LEHD-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. The ready-to-use caspase substrate provides an economic alternative for researchers who perform large amount of caspase assays. Cell Lysis Buffer (Cat. #1067-100, - 400) and 2X Reaction Buffer (Cat. #1068-20, -80) and DTT (Cat.# 1201-1) for caspase assays are also available separately.

Molecular Weight: 765.0

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 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 1 mM LEHD-AFC (50 μM 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 LEHD-dependent activity can be determined by comparing these results with the level of the uninduced control.

Purity: >98% by HPLC analysis.

Restrictions: For Research Use only

Publications

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Vu, Bortner, Cidlowski: "Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death." in: The Journal of biological chemistry, Vol. 276, Issue 40, pp. 37602-11, 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).

Ghatak, Misra, Toole: "Hyaluronan oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase/Akt cell survival pathway." in: The Journal of biological chemistry, Vol. 277, Issue 41, pp. 38013-20, 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).

Kawakami, Kawakami, Puri: "Intratumor administration of interleukin 13 receptor-targeted cytotoxin induces apoptotic cell death in human malignant glioma tumor xenografts." in: Molecular cancer therapeutics, Vol. 1, Issue 12, pp. 999-1007, 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).

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).

Lieman, Worley, Harbour: "Loss of Rb-E2F repression results in caspase-8-mediated apoptosis through inactivation of focal adhesion kinase." in: The Journal of biological chemistry, Vol. 280, Issue 11, pp. 10484-90, 2005 (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).

Wei, Wang, Topczewski et al.: "Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells." in: American journal of physiology. Endocrinology and metabolism, Vol. 291, Issue 2, pp. E275-81, 2006 (PubMed).

Lee, Cherla, Tesh: "Simultaneous induction of apoptotic and survival signaling pathways in macrophage-like THP-1 cells by Shiga toxin 1." in: Infection and immunity, Vol. 75, Issue 3, pp. 1291-302, 2007 (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).

Yuan, Fu, Wang et al.: "Voltage-dependent anion channel 1 is involved in endostatin-induced endothelial cell apoptosis." in: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 22, Issue 8, pp. 2809-20, 2008 (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).