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Caspase-9 Substrate LEHD-AFC

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Enzyme Activity Assay (EAA), Functional Studies (Func)
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Sequence Ac-Leu-Glu-His-Asp-AFC
Characteristics 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 for caspase assays are also available separately.
Purity > 98 % by HPLC
Molecular Weight 765.7 Da
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 LEHD-AFC (50 µM 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 LEHD-dependent 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

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Yano, Itoh, Kubota, Sendo, Koyama, Fujita, Saeki, Yuo, Oishi: "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

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

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

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

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

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

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

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

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

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

Tabata, Tabata, Grabowski, Bukowski, Ganapathi, Ganapathi: "Roles of NF-kappaB and 26 S proteasome in apoptotic cell death induced by topoisomerase I and II poisons in human nonsmall cell lung carcinoma." in: The Journal of biological chemistry, Vol. 276, Issue 11, pp. 8029-36, 2001

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

Ding, Lin, McGill, Juo, Zhu, Blenis, Yuan, Fisher: "Essential role for caspase-8 in transcription-independent apoptosis triggered by p53." in: The Journal of biological chemistry, Vol. 275, Issue 49, pp. 38905-11, 2001